Generic matrix for control nucleic acids

ABSTRACT

The present invention is in the field of in-vitro diagnostics. Within this field, it provides the amplification of at least a first target nucleic acid that may be present in at least one fluid sample using an internal control nucleic acid for qualitative and/or quantitative purposes and at least one external control nucleic acid in an aqueous buffer. It further provides an analytical system comprising an internal control nucleic acid for qualitative and/or quantitative purposes and at least one external control nucleic acid in an aqueous buffer.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. § 119 of EP Application No. 10195777.7 filed on Dec. 17, 2010. The entire disclosure of the above-referenced prior application is hereby incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention belongs to the field of in-vitro diagnostics. Within this field, it particularly concerns the isolation, amplification and detection of at least a first target nucleic acid that may be present in at least one fluid sample using an internal control nucleic acid for qualitative and/or quantitative purposes and at least one external control nucleic acid in an aqueous buffer.

BACKGROUND OF THE INVENTION

In the field of molecular diagnostics, the amplification of nucleic acids from numerous sources has been of considerable significance. Examples for diagnostic applications of nucleic acid amplification and detection are the detection of viruses such as Human Papilloma Virus (HPV), West Nile Virus (WNV) or the routine screening of blood donations for the presence of Human Immunodeficiency Virus (HIV), Hepatitis-B (HBV) and/or C Virus (HCV). Furthermore, said amplification techniques are suitable for bacterial targets such as mycobacteria, or the analysis of oncology markers.

The most prominent and widely-used amplification technique is Polymerase Chain Reaction (PCR). Other amplification reactions comprise, among others, the Ligase Chain Reaction, Polymerase Ligase Chain Reaction, Gap-LCR, Repair Chain Reaction, 3SR, NASBA, Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), and Qβ-amplification.

Automated systems for PCR-based analysis often make use of real-time detection of product amplification during the PCR process in the same reaction vessel. Key to such methods is the use of modified oligonucleotides carrying reporter groups or labels:

It has been shown that amplification and detection of more than one target nucleic acid in the same vessel is possible. This method is commonly termed “multiplex” amplification and requires different labels for distinction if real-time detection is performed.

It is mostly desirable or even mandatory in the field of clinical nucleic acid diagnostics to control the respective amplification using control nucleic acids with a known sequence, for qualitative (performance control) and/or quantitative (determination of the quantity of a target nucleic acid using the control as a reference) purposes. Given the diversity especially of diagnostic targets, comprising prokaryotic, eukaryotic as well as viral nucleic acids, and given the diversity between different types of nucleic acids such as RNA and DNA, control nucleic acids are usually designed in a specific manner.

In an in-vitro-diagnostic environment, assays based on nucleic acid amplification and detection are typically controlled externally, i.e. the respective control nucleic acid is processed in a vessel separate from the target nucleic acid. It can serve as a qualitative or a quantitative external control nucleic acid. In a qualitative setup, the control serves as a positive control for evaluating the validity of the amplification and detection reaction. In a quantitative setting, the control serves, alternatively or additionally, as a reference for determining the quantity or concentration of the target nucleic acid.

Fluid samples derived from biological fluids such as, for instance, human blood, often exhibit a significant complexity. Therefore, in order to mimic the fluid matrix of the biological samples possibly containing one or more target nucleic acids, external control nucleic acids used in in-vitro diagnostics usually have to be provided in essentially the same fluid matrix as said biological samples. For example, for the nucleic acid diagnostic analysis of human plasma, an external control nucleic is generally provided in normal human plasma (NHP, pooled plasma from several healthy donors), especially in order to comply with regulatory requirements (Guidance for Industry and FDA Staff—Assayed and Unassayed Quality Control Material, 2007; Section IV, B, 1. Matrix Effects).

The present invention provides a controlled amplification method using a different approach that displays various advantages.

SUMMARY OF THE INVENTION

The present invention provides processes for controlled amplification of a target nucleic acid, comprising:

-   -   a) providing in a reaction vessel an internal control nucleic         acid and a fluid sample, wherein the fluid sample may comprise a         target nucleic acid,     -   b) providing a solid support material to the reaction vessel for         a period of time and under conditions sufficient to permit         nucleic acids comprising the target nucleic acid and the         internal control nucleic acid to be immobilized on the solid         support material,     -   c) isolating the solid support material from other material         present in the reaction vessel in a separation station,     -   d) purifying the nucleic acids in the separation station and         washing the solid support material one or more times with a wash         buffer,     -   e) providing in a first reaction vessel the purified nucleic         acids and in a second reaction vessel an external control         nucleic acid in an aqueous buffer, wherein the aqueous buffer         lacks a fluid sample,     -   f) adding identical amplification reagents to each of the first         and second reaction vessels,     -   g) incubating the first and second reaction vessels under         identical conditions sufficient for amplification reactions to         occur, and     -   h) detecting results of the amplification reactions,         wherein the results of the amplification reactions are         indicative of the presence or absence of amplification of the         target nucleic acid, the internal control nucleic acid and the         external control nucleic acid.

In an embodiment, the process provides wherein the aqueous buffer has a pH between 6.0 and 12.0 and comprises: 1-100 mM Tris, 0.01-1 mM EDTA, 0.005-0.5% (w/v) Sodium Azide, and 1-200 mg/l Poly(rA) RNA.

In an embodiment, the process provides wherein the aqueous buffer has a pH of about 8 and comprises: 10 mM Tris, 0.1 mM EDTA, 0.05% (w/v) Sodium Azide, and 20 mg/l Poly(rA) RNA.

In an embodiment, the process provides wherein a negative control is provided in a third reaction vessel and is subjected to steps b) through h). Further, in an embodiment of the process the fluid sample is a clinical sample. In another embodiment, the process further comprises determining the quantity of the target nucleic acid.

The present invention also provide an analytical systems (440) for controlled amplification of a target nucleic acid comprising an amplification station comprising reaction vessels, the reaction vessels comprising amplification reagents, purified target nucleic acid and a purified internal control nucleic acid, and at least one external control nucleic acid in an aqueous buffer, wherein the aqueous buffer lacks a fluid sample, and wherein the at least one external control nucleic acid in an aqueous buffer is comprised in a different vessel than the purified target nucleic acid.

Another embodiment of the invention provides analytical systems further comprising a separation station comprising a solid support material, the separation station being constructed and arranged to separate and purify nucleic acids comprised in at least one fluid sample. In another embodiment the reaction vessels are arranged in an integral arrangement.

Another embodiment of the invention provides wherein the integral arrangement is a multiwell plate and the at least one external control nucleic acid in an aqueous buffer is comprised in a different well than the purified target nucleic acid. Additionally, the invention provides wherein the amplification station further comprises a negative control in a different vessel than the purified target nucleic acid and the external control nucleic acid in an aqueous buffer. Further, the invention provides wherein the negative control is the aqueous buffer.

An embodiment of the invention may further comprise one or more elements selected from the group consisting of: a detection module for detecting signals evoked by an analyte, a sealer, a storage module for reagents and/or disposables, and a control unit for controlling system components.

DESCRIPTION OF THE INVENTION

In a first aspect, the invention relates to a process for isolating and amplifying a target nucleic acid that may be present in at least one fluid sample, said process comprising the automated steps of:

-   a. adding an internal control nucleic acid to said fluid sample -   b. combining together a solid support material and said fluid sample     in a vessel for a period of time and under conditions sufficient to     permit nucleic acids comprising the target nucleic acid and the     internal control nucleic acid to be immobilized on the solid support     material -   c. isolating the solid support material from the other material     present in the fluid sample in a separation station -   d. purifying the nucleic acids in said separation station and     washing the solid support material one or more times with a wash     buffer -   e. contacting the purified target nucleic acid and the purified     internal control nucleic acid in at least a first vessel and at     least one external control nucleic acid in an aqueous buffer in at     least a second vessel with one or more amplification reagents -   f. incubating in said reaction vessels said purified target nucleic     acid, said purified internal control nucleic acid and said at least     one external control nucleic acid in an aqueous buffer with said one     or more amplification reagents for a period of time and under     conditions sufficient for an amplification reaction indicative of     the presence or absence of said target nucleic acid and said control     nucleic acids to occur     wherein the conditions for amplification in steps e. to f. are     identical for said purified target nucleic acid, said internal     control nucleic acid and said at least one external control nucleic     acid in an aqueous buffer.

The process described above is particularly advantageous since it does not involve any natural or complex media such as e.g. normal human plasma as a fluid matrix for the external control, but an aqueous buffer which is mostly significantly easier and cheaper to produce, to handle and to store. Moreover, since normal human plasma is derived from the blood of several individuals, its contents exhibit considerable variances. This includes the potential presence e.g. of inhibitors of amplification, or nuclease such as e.g. DNase activity.

In the former case, the presence of said inhibitors can jeopardize the normal human plasma's suitability for serving as a fluid matrix for an external control nucleic acid. The results of amplification and detection of such an external control nucleic acid are considerably likely to vary from lot to lot of said normal human plasma, or even from sample to sample.

In the case of a lot of normal human plasma showing e.g. significant DNase activity, the entire lot may have to be discarded since nucleic acids are not stable therein.

An aqueous buffer serving as a fluid matrix for an external control nucleic acid, as used in the present invention, overcomes these disadvantages. Since such a buffer is produced synthetically, the absence of amplification inhibitors or nucleases can be controlled relatively easily. Such undesired molecules might only enter said buffer as a result of contamination events, but the person skilled in the art is well aware of suitable measures to avoid contamination. Thus, in embodiments, the external control nucleic acids may even be stored in said aqueous buffer prior to being processed, which may be problematic in complex media such as normal human plasma. These and other reasons render the present invention particularly advantageous if the fluid sample is blood or blood plasma, especially human blood or blood plasma.

The following types of control nucleic acids are included in the process according to the invention:

-   -   Internal control nucleic:         -   acts as the full process control (i.e. nucleic acid             purification, amplification, and detection)         -   acts as a monitor for compromising effects on the target             reaction which are related to the composition of the             specific sample     -   External control nucleic acid in an aqueous buffer:         -   monitors the integrity of the assay-specific reagents         -   monitors the capability of the system to detect a             target-specific signal         -   need not act as a full process control and therefore does             not need to be manufactured within a sample matrix.

“Aqueous buffer” means any chemical buffer substance dissolved in water, and does not comprise biological buffers derived from natural sources, such as e.g. blood or blood plasma. Aqueous buffers particularly useful in the context of the invention are for example Tris, Tricine, HEPES, MOPS, citrate buffers such as e.g. sodium citrate or other buffers known to the skilled artisan. In an embodiment, the aqueous buffer is Tris. In another embodiment, the aqueous buffer is Tricine. For example, said aqueous buffer contains a chelating agent such as e.g. EDTA or EGTA, or others. By complex-binding, these agents coordinate cations that serve as cofactors for various enzymes that can damage nucleic acids, particularly nucleases that mostly depend on Mg2+. For example, the aqueous buffer described above contains EDTA. Furthermore, the aqueous buffer may contain a preserving agent. These agents are known by the skilled artisan. In an embodiment, the aqueous buffer described above contains sodium azide. In example said aqueous buffer further contains agents stabilizing the external control nucleic acid, for example macromolecules, such as for example poly(rA)RNA or glycogen. Further, the aqueous buffer described above has a neutral or alkaline pH, for example from a value of 6, 6.5, 7 or 7.5, to a value of 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12. For example, the aqueous buffer has a pH of about 8.

In an embodiment of the invention, the aqueous buffer mentioned above comprises the following constituents:

-   -   Tris: in a concentration from a value of 1, 2, 3, 4, 5, 6, 7, 8,         or 9 mM, to a value of 12.5, 15, 17.5, 20, 30, 40, 50, 60, 70,         80, 90, or 100 mM. For example, the concentration is about 10         mM.     -   EDTA: in a concentration from a value of 0.01, 0.02, 0.03, 0.04,         0.05, 0.06, 0.07, 0.08, or 0.09 mM, to a value of 0.125, 0.15,         0.175, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mM. For         example, the concentration is about 0.1 mM.     -   Sodium Azide: in a concentration from a value of 0.005, 0.01,         0.02, 0.03, or 0.04% (w/v), to a value of 0.06, 0.07, 0.08,         0.09, 0.1, or 0.5% (w/v). For example, the concentration is         about 0.05% (w/v).     -   Poly(rA)RNA: in a concentration from a value of 1, 2.5, 5, 7.5,         10, 12.5, 15, or 17.5 mg/l, to a value of 22.5, 25, 27.5, 30,         40, 50, 75, 100, 150, 175, or 200 mg/l. For example, the         concentration is about 20 mg/l.

Another considerable advantage is the independence of the choice of said aqueous buffer from the type of fluid sample, which is possible due to the presence of an internal control within said sample or samples in addition to said external control nucleic acid in an aqueous buffer. The fact that an internal control is present in the sample or samples abolishes the need for choosing a fluid matrix for the external control, wherein said fluid matrix mimics the fluid matrix of the sample or samples as closely as possible. The internal control nucleic acid as such is capable of controlling the potential presence or absence e.g. of inhibiting substances in the sample, such as for example PCR inhibitors possibly interfering with the analytic procedures. Thus, the external control nucleic acid need not be within a fluid matrix that closely resembles the fluid sample to be analyzed, but said fluid matrix may be any aqueous buffer such as a chemical aqueous buffer.

This circumstance is especially advantageous when preparing and analyzing a panel of nucleic acids from different sources such as e.g. clinical samples in different body fluids, for instance blood plasma, sputum or nasal swabs. In such a case, the external control nucleic acid can be provided in the same aqueous buffer instead of being provided in all of the fluid matrices blood plasma, sputum and nasal swab. If these different samples are prepared and analyzed in an integral arrangement of vessels such as a multiwell plate, even one well containing one external control nucleic acid in an aqueous buffer may be sufficient, e.g. if the target nucleic acid is the same in all samples, or if said external control nucleic acid is generic, i.e. suitable for different nucleic acids.

In addition to the internal and external control nucleic acids mentioned supra, it can also be advantageous to include a negative control in the process. The “negative control” is also an external control, however there is no nucleic acid included. This control is also termed “contamination control”, as a positive result in said control is indicative of a contamination of one or more assay components. In such a case, the respective test run is not considered valid. Thus, the negative control serves as a measure of contamination events on the system and therefore does not need to be manufactured within a sample matrix.

In an embodiment, the negative control is subjected to all steps of the process according to the invention following step a. In such an embodiment, the negative control serves as a full-process control for contamination, i.e. it is also suited to monitor potential contaminants introduced during the sample preparation procedure.

The negative control may essentially only consist of a fluid matrix such as an aqueous buffer. In an embodiment of the invention, the negative control is the same aqueous buffer as the aqueous buffer used for the external control nucleic acid. Again, there is no need to use a matrix similar or identical to the sample matrix, since in the process according to the invention an internal control nucleic acid is included. Thus, in a further embodiment, the negative control is water, for example distilled or deionized water.

The internal control nucleic acid mentioned above can be competitive, non-competitive or partially competitive.

A competitive internal control nucleic acid carries essentially the same primer binding sites as the target and thus competes for the same primers with the target. While this principle allows a good mimicry of the respective target nucleic acid due to their similar structure, it can lower the amplification efficiency with regard to the target nucleic acid or acids and thus lead to a less sensitive assay.

A non-competitive internal control nucleic acid has different primer binding sites than the target and thus binds to different primers. Advantages of such a setup comprise, among others, the fact that the single amplification events of the different nucleic acids in the reaction mixture can take place independently from each other without any competition effects. Thus, no adverse effects occur regarding the limit of detection of the assay as can be the case in a competitive setup.

Finally, in an amplification using a partially competitive setup the respective control nucleic acid and at least one of the target nucleic acids compete for the same primers, while at least one other target nucleic acid binds to different primers.

In a non-competitive setup, the internal control nucleic acid has a sequence different from any target sequences, in order not to compete for their primers and/or probes. For example, the sequence of the internal control nucleic acid is different from the other nucleic acid sequences in the fluid sample. As an example, if the fluid sample is derived from a human, the internal control nucleic acid does not have a sequence which also endogenously occurs within humans. The difference in sequence should thus be at least significant enough to not allow the binding of primers and/or probes to the respective endogenous nucleic acid or acids under stringent conditions and thus render the setup competitive. In order to avoid such interference, the sequence of the internal control nucleic acid used in the invention is derived from a source different from the origin of the fluid sample. For example, it is derived from a naturally occurring genome, for example a plant genome, further for example from a grape genome. In an embodiment, a nucleic acid derived from a naturally occurring genome is scrambled. As known in the art, “scrambling” means introducing base mutations in a sequence to a certain extent. For example, the sequence of the internal control nucleic acid used in the invention is substantially altered with respect to the naturally occurring gene it is derived from.

Another aspect of the invention relates to the process described supra, wherein at least a first and a second target nucleic acid are amplified in the same reaction vessel, thus allowing for the simultaneous amplification of a higher number of different target nucleic acids, since signals in different reaction vessels can be detected independently from each other. This multiplex reaction may take place in multiple reaction vessels, thereby multiplying the number of targets that may be amplified simultaneously using the same aqueous buffer for the external control nucleic acid and the same negative control.

In other cases, it may be desirable to amplify the first, but not the second target nucleic acid in the first reaction vessel, e.g. depending on the sample and/or the target nucleic acid or acids in question.

Therefore, a further embodiment of the invention is the process described above, wherein the second target nucleic acid is absent from the first reaction vessel.

Especially if a fluid sample is suspected to contain target nucleic acids from different organisms, or even the different organisms as such, or if it is not clear which of the different nucleic acids or organisms may be present in said sample, an advantageous embodiment of the invention is the process described above, wherein at least a first and a second target nucleic acid may be present in said sample, wherein said target nucleic acids are for example from different organisms.

As mentioned before, the method described above is useful for qualitatively or quantitatively controlling the amplification of one or more target nucleic acids.

Qualitative detection of a nucleic acid in a biological sample is crucial e.g. for recognizing an infection of an individual. Thereby, one important requirement for an assay for detection of e.g. a microbial infection is that false-negative or false-positive results be avoided, since such results would almost inevitably lead to severe consequences with regard to treatment of the respective patient. Thus, especially in PCR-based methods, a qualitative external or internal control nucleic acid is mostly added to the assay. Said control is particularly important for confirming the validity of a test result: At least in the case of a negative result with regard to the respective target nucleic acid, the control reaction has to perform reactive within given settings, i.e. the control nucleic acid must be detected, otherwise the test itself is considered to be inoperative. However, in a qualitative setup, said control does not necessarily have to be detected in case of a positive result. For qualitative tests, it is especially important that the sensitivity of the reaction is guaranteed and therefore strictly controlled. As a consequence the concentration of the qualitative control nucleic acid must be relatively low, so that even in a situation e.g. of slight inhibition the qualitative control nucleic acid is not detected and therefore the test is invalidated.

The use of an internal control nucleic acid bears the advantage of controlling the reaction performance in situ, i.e. for instance possible inhibiting agents that may have been introduced with the sample.

Thus, an aspect of the invention is the process described above, wherein the presence of an amplification product of said internal control nucleic acid is indicative of an amplification occurring in the vessel containing the purified target nucleic acid and the purified internal control nucleic acid, even in the absence of amplification products for said target nucleic acid.

On the other hand, if an external control nucleic acid in an aqueous buffer is used as a qualitative control, it monitors the principal capacity of the target to be amplified independently from the sample, thus providing a means to control the physical conditions under which the incubation is carried out. For example, in a PCR, the external control nucleic acid would not be amplified in case of an inappropriate profile, i.e. at inappropriate incubation temperatures and/or incubation times. A lack of amplification of said external control nucleic acid may also point to defects in the specific amplification reagents (such as specific primers) or non-specific amplification reagents (such as e.g. deoxynucleoside triphosphates or the respective DNA polymerase).

Thus, another aspect of the invention is the process described above, wherein the presence of an amplification product of said external control nucleic acid is indicative of an amplification occurring in the vessel containing the purified external nucleic acid, even in the absence of amplification products for said target nucleic acid.

On the other hand and in addition to mere detection of the presence or absence of a nucleic acid in a sample, it is often important to determine the quantity of said nucleic acid. As an example, stage and severity of a viral disease may be assessed on the basis of the viral load. Further, monitoring of any therapy requires information on the quantity of a pathogen present in an individual in order to evaluate the therapy's success. For a quantitative assay, it is necessary to introduce a quantitative standard nucleic acid serving as a reference for determining the absolute quantity of a target nucleic acid. Quantitation can be effectuated either by referencing to an external calibration or by implementing an internal quantitative standard.

Therefore, an aspect of the invention is the process described above, further comprising the following step:

-   -   g. determining the quantity of said target nucleic acid.

In the case of an external calibration, standard curves are created in separate reactions using known amounts of an identical or comparable nucleic acid, e.g. the at least one external control nucleic acid in an aqueous buffer mentioned above. The absolute quantity of a target nucleic acid is subsequently determined by comparison of the result obtained with the analyzed sample with said standard function. External calibration, however, has the disadvantage that a possible extraction procedure, its varied efficacy, and the possible and often not predictable presence of agents inhibiting the amplification and/or detection reaction are not reflected in the control.

This circumstance applies to any sample-related effects. Therefore, it might be the case that a sample is judged as negative due to an unsuccessful extraction procedure or other sample-based factors, whereas the target nucleic acid to be detected and quantified is actually present in the sample.

For these and other reasons, an internal control nucleic acid added to the test reaction itself is of advantage. When serving as a quantitative standard, said internal control nucleic acid has at least the following two functions in a quantitative test:

i) It monitors the validity of the reaction.

ii) It serves as reference in titer calculation thus compensating for effects of inhibition and controlling the preparation and amplification processes to allow a more accurate quantitation.

Therefore, in contrast to the qualitative internal control nucleic acid in a qualitative test which must be positive only in a target-negative reaction, the quantitative control nucleic acid in a quantitative test has two functions: reaction control and reaction calibration. Therefore it must be positive and valid both in target-negative and target-positive reactions.

It further has to be suited to provide a reliable reference value for the calculation of high nucleic acid concentrations. Thus, the concentration of an internal quantitative control nucleic acid needs to be relatively high.

In summary, the combination of an internal control nucleic acid and an external control nucleic acid in an aqueous buffer, as realized in the present invention, is highly advantageous. The combination not only combines the advantages set out above with regard to an external control nucleic acid alone or an internal control nucleic acid alone, but also leads to several synergistic effects:

-   -   Facilitation of trouble shooting in case of a failed experiment:         -   If e.g. only an internal or only an external control nucleic             acid is used, failure of their amplification only leads to             the conclusion that the run is invalid, but not why this is             the case. In contrast, if both types of controls are used,             and e.g. only the external control nucleic acid is             amplified, then it is likely that an inhibitor is present in             the sample, because the amplification reagents work on the             external control nucleic acid. This synergistic effect is             even enhanced in the embodiment, in which a negative control             is included, since then possible non-desired and thus             contaminating nucleic acids can be indicated by a positive             result in said negative control.     -   Enablement of the use of an aqueous buffer for the external         control nucleic acid:         -   As described further above, the inclusion of an internal             control nucleic acid within the fluid sample to be analyzed             abolishes the need to use a fluid matrix similar or             identical to the matrix of the fluid sample for the external             control nucleic acid. The fluid sample matrix does not have             to be mimicked because the internal control nucleic acid             acts as a full process control and is able to monitor             possible inhibitors in the fluid sample.     -   Regulatory requirements in clinical molecular diagnostics         demanding the provision of external control nucleic acids in         complex fluid matrices, as described above, may be complied with         even when using an external control nucleic acid in an aqueous         buffer as in the method of the present invention (Guidance for         Industry and FDA Staff—Assayed and Unassayed Quality Control         Material, 2007; Section IV, B, 1. Matrix Effects).

It can further be advantageous to subject the external control nucleic acid in an aqueous buffer to the procedure of sample preparation, i.e. isolation of nucleic acids. In these embodiments, a negative amplification result in said external control can be indicative of a defect in the sample preparation procedure, e.g. pointing towards a loss of nucleic acids.

Thus, an aspect of the invention is the process described above, wherein said at least one external control nucleic acid in an aqueous buffer is subjected to the steps following step a.

It is further possible to include both an external control nucleic acid subjected to said additional steps, and another external control nucleic acid being only subjected to the steps following step d, i.e. after sample preparation. In this embodiment, both advantages mentioned above can be combined.

Thus, an aspect of the invention is the process mentioned above, wherein at least one external control nucleic acid in an aqueous buffer is subjected to the steps following step a, and at least one other external control nucleic acid in an aqueous buffer is subjected only to the steps following step d.

Further advantageous are embodiments wherein said internal control nucleic acid is also added to said at least one external control nucleic acid in an aqueous buffer in step a or after step d. Among other advantages, this facilitates the workflow especially in automated setups, since the internal control nucleic acid may simply be added e.g. to all wells of a multiwell plate, containing one or more fluid samples and one or more external control nucleic acid in an aqueous buffer.

Therefore, an aspect of the invention is the process described above, further comprising:

in step a.: adding said internal control nucleic acid to at least one external control nucleic acid in an aqueous buffer.

In the sense of the invention, “purification”, “isolation” or “extraction” of nucleic acids relate to the following: Before nucleic acids may be analyzed in a diagnostic assay e.g. by amplification, they typically have to be purified, isolated or extracted from biological samples containing complex mixtures of different components. Often, for the first steps, processes are used which allow the enrichment of the nucleic acids. To release the contents of cells or viral particles, they may be treated with enzymes or with chemicals to dissolve, degrade or denature the cellular walls or viral particles. This process is commonly referred to as lysis. The resulting solution containing such lysed material is referred to as lysate. A problem often encountered during lysis is that other enzymes degrading the component of interest, e.g. deoxyribonucleases or ribonucleases degrading nucleic acids, come into contact with the component of interest during the lysis procedure. These degrading enzymes may also be present outside the cells or may have been spatially separated in different cellular compartments prior to lysis. As the lysis takes place, the component of interest becomes exposed to said degrading enzymes. Other components released during this process may e.g. be endotoxins belonging to the family of lipopolysaccharides which are toxic to cells and can cause problems for products intended to be used in human or animal therapy.

There is a variety of means to tackle the above-mentioned problem. It is common to use chaotropic agents such as guanidinium thiocyanate or anionic, cationic, zwitterionic or non-ionic detergents when nucleic acids are intended to be set free. It is also an advantage to use proteases which rapidly degrade the previously described enzymes or unwanted proteins. However, this may produce another problem as said substances or enzymes can interfere with reagents or components in subsequent steps.

Enzymes which can be advantageously used in such lysis or sample preparation processes mentioned above are enzymes which cleave the amide linkages in protein substrates and which are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W. H. Freeman and Company, San Francisco, Chapter 3). Proteases used in the prior art comprise alkaline proteases (WO 98/04730) or acid proteases (U.S. Pat. No. 5,386,024). A protease which has been widely used for sample preparation in the isolation of nucleic acids in the prior art is proteinase K from Tritirachium album (see e.g. Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) which is active around neutral pH and belongs to a family of proteases known to the person skilled in the art as subtilisins. Especially advantageous for the use in lysis or sample preparation processes mentioned above is the enzyme esperase, a robust protease that retains its activity at both high alkalinity and at high temperatures (EP 1 201 753).

In the sample preparation steps following the lysis step, the component of interest is further enriched. If the non-proteinaceous components of interest are e.g. nucleic acids, they are normally extracted from the complex lysis mixtures before they are used in a probe-based assay.

There are several methods for the purification of nucleic acids:

-   -   sequence-dependent or biospecific methods as e.g.:         -   affinity chromatography         -   hybridization to immobilized probes     -   sequence-independent or physico-chemical methods as e.g.:         -   liquid-liquid extraction with e.g. phenol-chloroform         -   precipitation with e.g. pure ethanol         -   extraction with filter paper         -   extraction with micelle-forming agents such as             cetyl-trimethyl-ammonium-bromide         -   binding to immobilized, intercalating dyes, e.g. acridine             derivatives         -   adsorption to silica gel or diatomic earths         -   adsorption to magnetic glass particles (MGP) or             organo-silane particles under chaotropic conditions

Particularly interesting for purification purposes is the adsorption of nucleic acids to a glass surface although other surfaces are possible. Many procedures for isolating nucleic acids from their natural environment have been proposed in recent years by the use of their binding behavior to glass surfaces. If unmodified nucleic acids are the target, a direct binding of the nucleic acids to a material with a silica surface is desirable because, among other reasons, the nucleic acids do not have to be modified, and even native nucleic acids can be bound. These processes are described in detail by various documents. In Vogelstein B. et al., Proc. Natl. Acad. USA 76 (1979) 615-9, for instance, a procedure for binding nucleic acids from agarose gels in the presence of sodium iodide to ground flint glass is proposed. The purification of plasmid DNA from bacteria on glass dust in the presence of sodium perchlorate is described in Marko M. A. et al., Anal. Biochem. 121 (1982) 382-387. In DE-A 37 34 442, the isolation of single-stranded M13 phage DNA on glass fiber filters by precipitating phage particles using acetic acid and lysis of the phage particles with perchlorate is described. The nucleic acids bound to the glass fiber filters are washed and then eluted with a methanol-containing Tris/EDTA buffer. A similar procedure for purifying DNA from lambda phages is described in Jakobi R. et al., Anal. Biochem. 175 (1988) 196-201. The procedure entails the selective binding of nucleic acids to glass surfaces in chaotropic salt solutions and separating the nucleic acids from contaminants such as agarose, proteins or cell residue. To separate the glass particles from the contaminants, the particles may be either centrifuged or fluids are drawn through glass fiber filters. This is a limiting step, however, that prevents the procedure from being used to process large quantities of samples. The use of magnetic particles to immobilize nucleic acids after precipitation by adding salt and ethanol is more advantageous and described e.g. in Alderton R. P. et al., S., Anal. Biochem. 201 (1992) 166-169 and PCT GB 91/00212. In this procedure, the nucleic acids are agglutinated along with the magnetic particles. The agglutinate is separated from the original solvent by applying a magnetic field and performing a wash step. After one wash step, the nucleic acids are dissolved in a Tris buffer. This procedure has a disadvantage, however, in that the precipitation is not selective for nucleic acids. Rather, a variety of solid and dissolved substances are agglutinated as well. As a result, this procedure can not be used to remove significant quantities of any inhibitors of specific enzymatic reactions that may be present. Magnetic, porous glass is also commercially available that contains magnetic particles in a porous, particular glass matrix and is covered with a layer containing streptavidin. This product can be used to isolate biological materials, e.g., proteins or nucleic acids, if they are modified in a complex preparation step so that they bind covalently to biotin. Magnetizable particular adsorbents proved to be very efficient and suitable for automatic sample preparation. Ferrimagnetic and ferromagnetic as well as superparamagnetic pigments are used for this purpose. For example magnetic glass particles and methods using them are those described in WO 01/37291. Particularly useful for the nucleic acid isolation in the context of the invention is the method according to R. Boom et al. (J Clin Microbiol. 28 (1990), 495-503).

The term “solid support material” comprises any of the solid materials mentioned above in connection with the immobilization of nucleic acids, e.g. magnetic glass particles, glass fibers, glass fiber filters, filter paper etc., while the solid support material is not limited to these materials.

Thus, an aspect of the invention is the process described above, wherein the solid support material comprises one or more of the materials selected from silica, metal, metal oxides, plastic, polymers and nucleic acids. In another embodiment of the invention, the solid support material is magnetic glass particles.

“Immobilize”, in the context of the invention, means to capture objects such as e.g. nucleic acids in a reversible or irreversible manner. Particularly, “immobilized on the solid support material”, means that the object or objects are associated with the solid support material for the purpose of their separation from any surrounding media, and can be recovered e.g. by separation from the solid support material at a later point. In this context, “immobilization” can e.g. comprise the adsorption of nucleic acids to glass or other suitable surfaces of solid materials as described supra. Moreover, nucleic acids can be “immobilized” specifically by binding to capture probes, wherein nucleic acids are bound to essentially complementary nucleic acids attached to a solid support by base-pairing. In the latter case, such specific immobilization leads to the predominant binding of target nucleic acids.

After the purification or isolation of the nucleic acids including the target nucleic acids from their natural surroundings, analysis may be performed e.g. via the amplification described supra.

The “first target nucleic acid” and the “second target nucleic acid” are different nucleic acids.

A “fluid sample” is any fluid material that can be subjected to a diagnostic assay targeting nucleic acids and can be derived from a biological source. In an embodiment of the process described above, the fluid sample is a clinical sample. Also for example, said fluid sample is derived from a human and is a body liquid. In an embodiment of the invention, the fluid sample is human blood, urine, sputum, sweat, swab, pipettable stool, or spinal fluid. Further for example, the fluid sample is human blood or human blood plasma.

The term “reaction vessel” or “vessel” comprises, but is not limited to, tubes or the wells of plates such as microwell, deepwell or other types of multiwell plates, in which a reaction for the analysis of the fluid sample such as e.g. reverse transcription or a polymerase chain reaction takes place. The outer limits or walls of such vessels are chemically inert such that they do not interfere with the analytical reaction taking place within. For example, the isolation of the nucleic acids as described above is also carried out in a multiwell plate, for example in a deepwell plate.

In some embodiments, the vessel in step b. of the process described above, in which the fluid sample is combined together with a solid support material, is different from the first reaction vessel and the second reaction vessel in step e., in which the nucleic acids are contacted with amplification reagents. Since in these embodiments the purification and amplification of the target nucleic acid are performed in different vessels, e.g. multiwell plates adapted for their respective purpose may be used, such as for instance a deepwell plate for purification and a multiwell plate with smaller vessels for amplification.

In other embodiments, the vessel from step b. is the same as the first reaction vessel in step e, meaning that the steps for purification and subsequent amplification of the target nucleic acid are carried out within the same vessel. Such embodiments bear the advantage that no more pipetting or similar transferring step is required between purification and amplification.

Multiwell plates in analytical systems allow parallel separation and analyzing or storage of multiple samples. Multiwell plates may be optimized for maximal liquid uptake, or for maximal heat transfer. An exemplary multiwell plate for use in the context of the present invention is optimized for incubating or separating an analyte in an automated analyzer. For example, the multiwell plate is constructed and arranged to contact a magnetic device and/or a heating device.

Said multiwell plate, which is interchangeably termed “processing plate” in the context of the invention, comprises:

-   -   a top surface comprising multiple vessels with openings at the         top arranged in rows. The vessels comprise an upper part, a         center part and a bottom part. The upper part is joined to the         top surface of the multiwell plate and comprises two longer and         two shorter sides. The center part has a substantially         rectangular cross-section with two longer sides and two shorter         sides;     -   two opposing shorter and two opposing longer side walls and     -   a base, wherein said base comprises an opening constructed and         arranged to place the multiwell plate in contact with said         magnetic device and/or a heating device.

In an embodiment of the multiwell plate, adjacent vessels within one row are joined on the longer side of said almost rectangular shape.

For example, the multiwell plate comprises a continuous space which is located between adjacent rows of vessels. Said continuous space is constructed and arranged to accommodate a plate-shaped magnetic device. In an embodiment, the bottom part of the vessels comprises a spherical bottom. In another embodiment, the bottom part of said vessels comprises a conical part located between said central part and said spherical bottom.

In an embodiment, the top surface comprises ribs, wherein said ribs surround the openings of the vessels. For example, one shorter side of said upper part of the vessels comprises a recess, said recess comprising a bent surface extending from the rib to the inside of the vessel.

Furthermore, in an embodiment, the vessels comprise a rounded inside shape.

For fixation to the processing or incubation stations, the base for example comprises a rim comprising recesses. Latch clips on a station of an analyzer can engage with said recesses to fix the plate on a station.

In an embodiment, the vessels comprise an essentially constant wall thickness.

An exemplary processing plate (101) in the context of the present invention is a 1-component plate. Its top surface (110) comprises multiple vessels (103) (FIG. 5, FIG. 6). Each vessel has an opening (108) at the top and is closed at the bottom end (112). The top surface (110) comprises ribs (104) which can be elevated relative to the top surface (110) and surround the openings (108) of the vessels (103). This prevents contamination of the contents of the vessels (103) with droplets of liquid that may fall onto the top surface (110) of the plate (101). Views of an exemplary process plate are shown in FIGS. 3 to 8.

The footprint of the processing plate (101) may comprise a length and width of the base corresponding to ANSI SBS footprint format. For example, the length is 127.76 mm+/−0.25 mm, and the width is 85.48 mm+/−0.25 mm. Thus, the plate (101) has two opposing shorter side walls (109) and two opposing longer side walls (118). The processing plate (101) comprises form locking elements (106) for interacting with a handler (500, FIG. 12). The processing plate (101) can be gripped, transported and positioned quickly and safely at high speed while maintaining the correct orientation and position. Further, the form locking elements (106) for gripping are located within the upper central part, for example the upper central third of the processing plate (101). This has the advantage that a potential distortion of the processing plate (101) has only a minor effect on the form locking elements (106) and that the handling of the plate (101) is more robust.

The processing plate (101) may comprise hardware-identifiers (102) and (115). The hardware identifiers (102) and (115) are unique for the processing plate (101) and different from hardware identifiers of other consumables used in the same system. The hardware identifiers (102, 115) may comprise ridges (119) and/or recesses (125) on the side walls of the consumables, wherein said pattern of ridges (119) and/or recesses (125) is unique for a specific type of consumable, for example the processing plate (101). This unique pattern is also referred to herein as a unique “surface geometry”. The hardware-identifiers (102, 115) ensure that the user can only load the processing plate (101) into the appropriate stacker position of an analytical instrument in the proper orientation. On the sides of processing plate (101), guiding elements (116) and (117) are comprised (FIG. 3, FIG. 4). They prevent canting of the processing plate (101). The guiding elements (116, 117) allow the user to load the processing plates (101) with guiding elements (116, 117) as a stack into an analytical instrument which is then transferred vertically within the instrument in a stacker without canting of the plates.

The center part (120) of the vessels (103) has an almost rectangular cross section (FIG. 6, FIG. 7). They are separated along the longer side (118) of the almost rectangular shape by a common wall (113) (FIG. 3). The row of vessels (103) formed thereby has the advantage that despite the limited space available, they have a large volume, for example 0.4 ml. Another advantage is that because of the essentially constant wall thickness, the production is very economical. A further advantage is that the vessels (103) strengthen each other and, thus, a high stability of the shape can be obtained.

Between the rows of vessels (103), a continuous space (121) is located (FIG. 6, FIG. 7). The space (121) can accommodate magnets (202, 203) or heating devices (128) (FIG. 11). These magnets (202, 203) and heating devices (128) are for example solid devices. Thus, magnetic particles (216) comprised in liquids (215) which can be held in the vessels (103) can be separated from the liquid (215) by exerting a magnetic field on the vessels (103) when the magnets (202, 203) are brought into proximity of the vessels (103). Or the contents of the vessels (103) can be incubated at an elevated, controlled temperature when the processing plate (101) is placed on the heating device (128). Since the magnets (202, 203) or heating devices (128) can be solid, a high energy density can be achieved. The almost rectangular shape of the central part (120) of the vessels (103) (FIG. 10) also optimizes the contact between the vessel wall (109) and a flat shaped magnet (202) or heating device (128) by optimizing the contact surface between vessel (103) and magnet (202) or heating device (128) and thus enhancing energy transfer into the vessel (103).

In the area of the conical bottom (111) of the vessels, the space (121) is even more pronounced and can accommodate further magnets (203). The combination of the large magnets (202) in the upper area and the smaller magnets (203) in the conical area of the vessels allows separation of magnetic particles (216) in larger or small volumes of liquid (215). The small magnets (203), thus, make it easier to sequester the magnetic particles (216) during eluate pipetting. This makes it possible to pipette the eluate with minimal loss by reducing the dead volume of the magnetic particle (216) pellet. Furthermore, the presence of magnetic particles (216) in the transferred eluate is minimized.

At the upper end of the vessels (103), one of the shorter side walls (109) of the vessel (103) comprises a reagent inlet channel (105) which extends to the circumferential rib (104) (FIGS. 3, 4, 7). The reagents are pipetted onto the reagent inlet channel (105) and drain off the channel (105) into the vessel (103). Thus, contact between the pipet needle or tip (3, 4) and liquid contained in the vessel is prevented. Furthermore, splashes resulting from liquid being directly dispensed into another liquid (215) contained in the vessels (103), which may cause contamination of the pipet needle or tip (3, 4) or neighboring vessels (103) is prevented. Sequential pipetting onto the reagent inlet channel (105) of small volumes of reagents followed by the largest volume of another reagent ensures that the reagents which are only added in small amounts are drained completely into the vessel (103). Thus, pipetting of small volumes of reagents is possible without loss of accuracy of the test to be performed.

On the inside, on the bottom of the vessels (111, 112), the shape becomes conical (111) and ends with a spherical bottom (112) (FIG. 6, FIG. 7). The inside shape of the vessel (114), including the rectangular center part (120), is rounded. The combination of spherical bottom (112), rounded inside shape (114), conical part (111) and refined surface of the vessels (103) leads to favorable fluidics which facilitate an effective separation and purification of analytes in the processing plate (101). The spherical bottom (112) allows an essentially complete use of the separated eluate and a reduction of dead-volume which reduces the carryover of reagents or sample cross-contamination.

The rim on the base (129) of the processing plate (101) comprises recesses (107) for engagement with latch clips (124) on the processing station (201) or heating device (128) or analytical instrument (126) (FIG. 5, FIG. 9). The engagement of the latch clips (124) with the recesses (107) allows positioning and fixation of the processing plate (101) on the processing station (201). The presence of the recesses (107) allows the latch force to act on the processing plate (101) almost vertically to the base (129). Thus, only small forces acting sideways can occur. This reduces the occurrence of strain, and, thus, the deformation of the processing plate (101). The vertical latch forces can also neutralize any deformations of the processing plate (101) leading to a more precise positioning of the spherical bottoms (111) within the processing station (201). In general, the precise interface between the processing plate (101) and the processing station (201) or heating device (128) within an analyzer reduces dead-volumes and also reduces the risk of sample cross-contamination.

A “separation station” is a device or a component of an analytical system allowing for the isolation of the solid support material from the other material present in the fluid sample. Such a separation station can e.g. comprise, but is not limited to, a centrifuge, a rack with filter tubes, a magnet, or other suitable components. In an embodiment of the invention, the separation station comprises one or more magnets. For example, one or more magnets are used for the separation of magnetic particles, for example magnetic glass particles, as a solid support. If, for example, the fluid sample and the solid support material are combined together in the wells of a multiwell plate, then one or more magnets comprised by the separation station can e.g. be contacted with the fluid sample itself by introducing the magnets into the wells, or said one or more magnets can be brought close to the outer walls of the wells in order to attract the magnetic particles and subsequently separate them from the surrounding liquid.

In an embodiment, the separation station is a device that comprises a multiwell plate comprising vessels with an opening at the top surface of the multiwell plate and a closed bottom. The vessels comprise an upper part, a center part and a bottom part, wherein the upper part is joined to the top surface of the multiwell plate and for example comprises two longer and two shorter sides. The center part has a substantially rectangular cross-section with two longer sides, wherein said vessels are aligned in rows. A continuous space is located between two adjacent rows for selectively contacting at least one magnet mounted on a fixture with the side walls in at least two Z-positions. The device further comprises a magnetic separation station comprising at least one fixture. The fixture comprises at least one magnet generating a magnetic field. A moving mechanism is present which vertically moves said at least one fixture comprising at least one magnet at least between first and second positions with respect to the vessels of the multiwell plate. For example, said at least two Z-positions of the vessels comprise the side walls and the bottom part of said vessels. The magnetic field of said at least one magnet for example draws the magnetic particles to an inner surface of the vessel adjacent to said at least one magnet when said at least one magnet is in said first position. The effect of said magnetic field is less when said at least one magnet is in said second position than when said at least one magnet is in said first position. For example, the fixture comprising said at least one magnet comprises a frame. The vessels may have features as described above in the context of multiwell plate/processing plate. One such feature is that at least a part of said vessels has a substantially rectangular cross-section orthogonal to the axis of said vessels.

In said first position, said at least one magnet is adjacent to said part of said vessels. Adjacent is understood to mean either in close proximity such as to exert a magnetic field on the contents of the vessel, or in physical contact with the vessel.

The separation station comprises a frame to receive the multiwell plate, and latch-clips to attach the multiwell plate. For example, the separation station comprises two types of magnets. This exemplary embodiment is further described below.

A second embodiment is described below, which comprises a spring which exerts a pressure on the frame comprising the magnets such that the magnets are pressed against the vessels of the multiwell plate.

The first magnets may be constructed and arranged to interact with vessels of a multiwell plate for exerting a magnetic field on a large volume of liquid comprising magnetic particles held in said vessels. Said second magnets may be constructed and arranged to interact with vessels of a multiwell plate for exerting a magnetic field on a small volume of liquid comprising magnetic particles held in said vessels. Said first and second magnets can be moved to different Z-positions.

Useful in the context of the present invention and said separation station is further a method of isolating and purifying a nucleic acid. The method comprises the steps of binding a nucleic acid to magnetic particles in a vessel of a multiwell plate. The vessel comprises an upper opening, a central part and a bottom part. The bound material is then separated from unbound material contained in a liquid when the major part of the liquid is located above the section where the conical part of the vessel is replaced by the central part with the rectangular shape, by moving a magnet from a second position to a first position and, in said first position, applying a magnetic field to the central part and, optionally, additionally applying a magnetic field to the bottom part of said vessel. The magnetic particles can optionally be washed with a washing solution. A small volume of liquid, wherein the major part of the liquid is located below the section where the conical part of the vessel is replaced by the central part with the rectangular shape is separated from said magnetic particles by selectively applying a magnetic field to the bottom part of said vessel.

Useful in the context of the present invention is also a magnetic separation station for separating a nucleic acid bound to magnetic particles, said separation station comprising first magnets which are constructed and arranged to interact with vessels of a multiwell plate for exerting a magnetic field on a large volume of liquid comprising magnetic particles held in said vessels, and second magnets constructed and arranged to interact with vessels of a multiwell plate for exerting a magnetic field on a small volume of liquid comprising magnetic particles held in said vessels, and wherein said first and second magnets can be moved to different Z-positions. Embodiments of the magnetic separation station are described herein.

An embodiment of a separation station (201) useful for the present invention is described below. The first embodiment of said separation station (201) comprises at least two types of magnets (202, 203). The first, long type of magnet (202) is constructed and arranged to fit into the space (121) of the processing plate (101). Magnet (202), thus, exerts a magnetic field on the liquid (215) in the vessel (103) to sequester magnetic particles (216) on the inside of the vessel wall. This allows separation of the magnetic particles (216) and any material bound thereto and the liquid (215) inside the vessel (103) when a large volume of liquid (215) is present. Magnet (202) has an elongated structure and is constructed and arranged to interact with the essentially rectangular central part (120) of the vessel. Thus, magnet (202) is used when the major part of the liquid (215) is located above the section where the conical part (111) of the vessel (103) is replaced by the central part (120) with the rectangular shape. As shown in FIG. 10, the construction of the magnets (202) comprises fixtures (204, 204 a) comprising magnets (202) which fit into the space (121) between the rows of vessels (103) in the processing plate (101). Another embodiment of magnets (202) comprises magnets (202) arranged on fixtures (204, 204 a). The magnets (203) of the separation station (201) are smaller, and can interact with the conical part (111) of the vessel (103). This is shown in FIG. 10. Magnets (203) are arranged on a base (205) which can be moved into the space (121) of the processing plate (101). Each magnet (202, 203) is constructed to interact with two vessels (103) in two adjacent rows. In an embodiment, the processing plate (101) has 6 rows of 8 vessels (103). A separation station (201) which can interact with the processing plate (101) has three fixtures (204, 204 a) comprising magnets (202) and four bases (205) comprising magnets (203). An embodiment is also included wherein the separation station has four magnetic fixtures (204, 204 a) comprising magnets (202) and three magnetic bases (205) comprising magnets (203).

The magnets (202, 203) are movable. The separation station (201) comprises a mechanism to move the fixtures (204, 204 a) and the bases (205). All fixtures (204, 204 a) are interconnected by a base (217) and are, thus, moved coordinately. All magnets (203) are joined to one base (218) and are, thus, moved coordinately. The mechanism for moving the magnetic plates (202) and (203) is constructed and arranged to move the two types of magnetic plates (202, 203) to a total of four end positions:

In FIG. 10 a-c, the magnets (203) are located in proximity of the conical part of the vessels (103) of the processing plate (101). This is the uppermost position of magnets (203), and is the separation position. In this Figure, the magnets (202) are located in the lowermost position. They are not involved in separation when they are in this position.

In an embodiment shown in FIG. 10, the base (217) of magnets (202) is connected to a positioning wheel (206). The base (217) comprises a bottom end (207) which is flexibly in contact with a connecting element (208) by a moving element (209). Said moving element is constructed and arranged to move the connecting element (208) along a rail (212) from one side to the other. Said moving element (209) is fixed to the connecting element (208) with a pin (220). Said connecting element (208) is fixed to the positioning wheel (206) by screw (210). Connecting element (208) is also connected to axis (211). Said connecting element (208) can be a rectangular plate. As the positioning wheel (206) moves eccentrically, around an axis (211), such that the screw (210) moves from a point above the eccentric axis to a point below the eccentric axis, moving element (209) and the bottom end (207) of the base (204) with the magnets (202) attached thereto are moved from the uppermost position to the lowermost position. The base (218) is mounted on a bottom part (219) and is connected, at its lower end, with pin (213) to a moving element (214), which can be a wheel, which interacts with the positioning wheel (206). When the positioning wheel (206) rotates around the axis (211), wheel (214) moves along positioning wheel (206). If the wheel (214) is located on a section of positioning wheel (206) where the distance from the axis (211) is short, the magnets (203) are in their lowermost position. When wheel (214) is located on a section of positioning wheel (206) where the distance from the axis (211) is at a maximum, the magnets (203) are in their uppermost position. Thus, in an embodiment of the first embodiment of the separation station, the location of the magnets (203) is controlled by the shape of the positioning wheel (206). When moving element (209) moves along the central, rounded upper or lower part (212 a) of rail (212), the small type of magnets (203) are moved up and down. When the moving element (209) is located on the side (212 b) of bottom end (207) and moves up or down, the magnets (202) are moved up- or downwards. The positioning wheel can be rotated by any motor (224).

In an embodiment, a spring (225) is attached to the base (222) of the separation station and the base (218) of magnets (203) to ensure that magnets (203) are moved into the lowermost position when they are moved downwards.

The term “pin” as used herein relates to any fixation element, including screws or pins.

In a second embodiment, the separation station (230) comprises at least one fixture (231) comprising at least one magnet (232), for example a number of magnets equal to a number of vessels (103) in a row (123). For example, the separation station (230) comprises a number of fixtures (231) equal to the number of rows (123) of the multiwell plate (101) hereinbefore described. Further, six fixtures (231) are mounted on the separation station (230). At least one magnet (232) is mounted on one fixture (231). For example, the number of magnets (232) equals the number of vessels (103) in one row (123). Further, eight magnets (232) are mounted on one fixture (231). Further, one type of magnet (232) is comprised on said fixture (231). Further, the magnet (232) is mounted on one side oriented towards the vessels with which the magnet interacts.

The fixture (231) is mounted on a base (233). For example, said mount is flexible. The base (233) comprises springs (234) mounted thereon. The number of springs (234) is at least one spring per fixture (231) mounted on said base (233). The base further comprises a chamfer (236) which limits the movement of the spring and, consequently, the fixture (231) comprising the magnets (232). For example, any one of said springs (234) is constructed and arranged to interact with a fixture (231). For example, said spring (234) is a yoke spring. Said interaction controls the horizontal movement of the fixtures (231). Furthermore, the separation station (230) comprises a frame (235). The base (233) with fixtures (231) is connected to the frame (235) by a moving mechanism as described hereinbefore for the magnets (232) of the first embodiment.

For example, said base (233) and fixture (231) is constructed and arranged to move vertically (in Z-direction).

The multiwell plate (101) hereinbefore described is inserted into the separation station (230). The fixture (231) comprising the magnets (232) is moved vertically. Any one fixture (232) is, thus, moved into a space (121) between two rows (123) of vessels (103). The vertical movement brings the magnets (232) mounted on a fixture (231) into contact with the vessels (103). The Z-position is chosen depending on the volume of liquid (215) inside the vessels (103). For large volumes, the magnets (232) contact the vessels (103) in a center position (120) where the vessels (103) are of an almost rectangular shape. For small volumes of liquid (215) where the major pail of the liquid (215) is located below the center part (120) of the vessels (103), the magnets (232) may contact the conical part (111) of the vessels (103).

A spring is attached to the base (233) of any one frame (231) (FIG. 9 a), b)). The spring presses the magnets (232) against the vessels (103). This ensures a contact between magnets (232) and vessels (103) during magnetic separation. For example, the magnet (232) contacts the vessel (103) on the side wall (109) located underneath the inlet (105). This has the advantage that liquid which is added by pipetting flows over the sequestered magnetic particles and ensures that particles are resuspended and that all samples in all vessels are treated identically.

This embodiment is particularly suited to separate a liquid (215) comprised in a multiwell plate (101) as hereinbefore described, from magnetic particles (216) when different levels of liquid (215) are contained in the vessels (103) of said multiwell plate (101).

A “wash buffer” is a fluid that is designed to remove undesired components, especially in a purification procedure. Such buffers are well known in the art. In the context of the purification of nucleic acids, the wash buffer is suited to wash the solid support material in order to separate the immobilized nucleic acid from any unwanted components. The wash buffer may, for example, contain ethanol and/or chaotropic agents in a buffered solution or solutions with an acidic pH without ethanol and/or chaotropic agents as described above. Often the washing solution or other solutions are provided as stock solutions which have to be diluted before use.

The washing in the process according to the invention requires a more or less intensive contact of the solid support material and the nucleic acids immobilized thereon with the wash buffer. Different methods are possible to achieve this, e.g. shaking the wash buffer with the solid support material in or along with the respective vessel or vessels. Another advantageous method is aspirating and dispensing the suspension comprising wash buffer and solid support material one or more times. This method may be carried out using a pipet, wherein said pipet comprises a disposable pipet tip into which said suspension is aspirated and from which it is dispensed again. Such a pipet tip can be used several times before being discarded and replaced. Disposable pipet tips useful for the invention for example have a volume of at least 10 μl at least 15 μl, at least 100 μl, at least 500 μl, of at least 1 ml, or of about 1 ml. Pipets used in the context of the invention can also be pipetting needles.

Thus, an aspect of the invention is the process described above, wherein said washing in step d. comprises aspirating and dispensing the wash buffer comprising the solid support material.

For downstream processing of the isolated nucleic acids, it can be advantageous to separate them from the solid support material before subjecting them to amplification.

Therefore, an aspect of the invention is the process described above, wherein said process further comprises in step d. the step of eluting the purified nucleic acids from the solid support material with an elution buffer after washing said solid support material.

An “elution buffer” in the context of the invention is a suitable liquid for separating the nucleic acids from the solid support. Such a liquid may e.g. be distilled water or aqueous salt solutions, such as e.g. Tris buffers like Tris HCl, or HEPES, or other suitable buffers known to the skilled artisan. The pH value of such an elution buffer is for example alkaline or neutral. Said elution buffer may contain further components such as e.g. chelators like EDTA, which stabilizes the isolated nucleic acids by inactivation of degrading enzymes.

The elution may be carried out at elevated temperatures, such that an embodiment of the invention is the process described above, wherein step d. is carried out at a temperature between 70° C. and 90° C., for example at a temperature of 80° C.

“Amplification reagents”, in the context of the invention, are chemical or biochemical components that enable the amplification of nucleic acids. Such reagents comprise, but are not limited to, nucleic acid polymerases, buffers, mononucleotides such as nucleoside triphosphates, oligonucleotides e.g. as oligonucleotide primers, salts and their respective solutions, detection probes, dyes, and more.

As is known in the art, a “nucleoside” is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines.

“Nucleotides” are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′-, 3′- or 5′-hydroxyl moiety of the sugar. A nucleotide is the monomeric unit of an “oligonucleotide”, which can be more generally denoted as an “oligomeric compound”, or a “polynucleotide”, more generally denoted as a “polymeric compound”. Another general expression for the aforementioned is deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

According to the invention, an “oligomeric compound” is a compound consisting of “monomeric units” which may be nucleotides alone or non-natural compounds (see below), more specifically modified nucleotides (or nucleotide analogs) or non-nucleotide compounds, alone or combinations thereof.

“Oligonucleotides” and “modified oligonucleotides” (or “oligonucleotide analogs”) are subgroups of oligomeric compounds. In the context of this invention, the term “oligonucleotide” refers to components formed from a plurality of nucleotides as their monomeric units. The phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Oligonucleotides and modified oligonucleotides (see below) useful for the invention may be synthesized as principally described in the art and known to the expert in the field. Methods for preparing oligomeric compounds of specific sequences are known in the art, and include, for example, cloning and restriction of appropriate sequences and direct chemical synthesis. Chemical synthesis methods may include, for example, the phosphotriester method described by Narang S. A. et al., Methods in Enzymology 68 (1979) 90-98, the phosphodiester method disclosed by Brown E. L., et al., Methods in Enzymology 68 (1979) 109-151, the phosphoramidite method disclosed in Beaucage et al., Tetrahedron Letters 22 (1981) 1859, the H-phosphonate method disclosed in Garegg et al., Chem. Scr. 25 (1985) 280-282 and the solid support method disclosed in U.S. Pat. No. 4,458,066.

In the process described above, the oligonucleotides may be chemically modified, i.e. the primer and/or the probe comprise a modified nucleotide or a non-nucleotide compound. The probe or the primer is then a modified oligonucleotide.

“Modified nucleotides” (or “nucleotide analogs”) differ from a natural nucleotide by some modification but still consist of a base, a pentofuranosyl sugar, a phosphate portion, base-like, pentofuranosyl sugar-like and phosphate-like portion or combinations thereof. For example, a label may be attached to the base portion of a nucleotide whereby a modified nucleotide is obtained. A natural base in a nucleotide may also be replaced by e.g. a 7-deazapurine whereby a modified nucleotide is obtained as well.

A “modified oligonucleotide” (or “oligonucleotide analog”), belonging to another specific subgroup of oligomeric compounds, possesses one or more nucleotides and one or more modified nucleotides as monomeric units. Thus, the term “modified oligonucleotide” (or “oligonucleotide analog”) refers to structures that function in a manner substantially similar to oligonucleotides and can be used interchangeably in the context of the present invention. From a synthetical point of view, a modified oligonucleotide (or an oligonucleotide analog) can for example be made by chemical modification of oligonucleotides by appropriate modification of the phosphate backbone, ribose unit or the nucleotide bases (Uhlmann and Peyman, Chemical Reviews 90 (1990) 543; Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134). Representative modifications include phosphorothioate, phosphorodithioate, methyl phosphonate, phosphotriester or phosphoramidate inter-nucleoside linkages in place of phosphodiester internucleoside linkages; deaza- or azapurines and -pyrimidines in place of natural purine and pyrimidine bases, pyrimidine bases having substituent groups at the 5 or 6 position; purine bases having altered substituent groups at the 2, 6 or 8 positions or 7 position such as 7-deazapurines; bases carrying alkyl-, alkenyl-, alkynyl or aryl-moieties, e.g. lower alkyl groups such as methyl, ethyl, propyl, butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, or aryl groups like phenyl, benzyl, naphtyl; sugars having substituent groups at, for example, their 2′ position; or carbocyclic or acyclic sugar analogs. Other modifications consistent with the spirit of this invention are known to those skilled in the art. Such modified oligonucleotides (or oligonucleotide analogs) are best described as being functionally interchangeable with, yet structurally different from, natural oligonucleotides. In more detail, exemplary modifications are disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134 or WO 02/12263. In addition, modification can be made wherein nucleoside units are joined via groups that substitute for the internucleoside phosphate or sugar phosphate linkages. Such linkages include those disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134. When other than phosphate linkages are utilized to link the nucleoside units, such structures have also been described as “oligonucleosides”.

A “nucleic acid” as well as the “target nucleic acid” is a polymeric compound of nucleotides as known to the expert skilled in the art. “Target nucleic acid” is used herein to denote a nucleic acid in a sample which should be analyzed, i.e. the presence, non-presence and/or amount thereof in a sample should be determined.

The term “primer” is used herein as known to the expert skilled in the art and refers to oligomeric compounds, primarily to oligonucleotides, but also to modified oligonucleotides that are able to prime DNA synthesis by a template-dependent DNA polymerase, i.e. the 3′-end of the e.g. primer provides a free 3′-OH group whereto further nucleotides may be attached by a template-dependent DNA polymerase establishing 3′- to 5′-phosphodiester linkage whereby deoxynucleoside triphosphates are used and whereby pyrophosphate is released.

A “probe” also denotes a natural or modified oligonucleotide. As known in the art, a probe serves the purpose to detect an analyte or amplificate. In the case of the process described above, probes can be used to detect the amplificates of the target nucleic acids. For this purpose, probes typically carry labels.

“Labels”, often referred to as “reporter groups”, are generally groups that make a nucleic acid, in particular oligonucleotides or modified oligonucleotides, as well as any nucleic acids bound thereto distinguishable from the remainder of the sample (nucleic acids having attached a label can also be termed labeled nucleic acid binding compounds, labeled probes or just probes). Exemplary labels according to the invention are fluorescent labels, which are e.g. fluorescent dyes such as a fluorescein dye, a rhodamine dye, a cyanine dye, and a coumarin dye. Exemplary fluorescent dyes according to the invention are FAM, HEX, JA270, CAL635, Coumarin343, Quasar705, Cyan500, CY5.5, LC-Red 640, LC-Red 705.

In the context of the invention, any primer and/or probe may be chemically modified, i.e. the primer and/or the probe comprise a modified nucleotide or a non-nucleotide compound. The probe or the primer is then a modified oligonucleotide.

An exemplary method of nucleic acid amplification is the Polymerase Chain Reaction (PCR) which is disclosed, among other references, in U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188. PCR typically employs two or more oligonucleotide primers that bind to a selected nucleic acid template (e.g. DNA or RNA). Primers useful for nucleic acid analysis include oligonucleotides capable of acting as a point of initiation of nucleic acid synthesis within the nucleic acid sequences of the target nucleic acids. A primer can be purified from a restriction digest by conventional methods, or it can be produced synthetically. The primer is for example single-stranded for maximum efficiency in amplification, but the primer can be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double stranded nucleic acids is by heating. A “thermostable polymerase” is a polymerase enzyme that is heat stable, i.e., it is an enzyme that catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded template nucleic acids. Generally, the synthesis is initiated at the 3′ end of each primer and proceeds in the 5′ to 3′ direction along the template strand. Thermostable polymerases have e.g. been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, polymerases that are not thermostable also can be employed in PCR assays provided the enzyme is replenished.

If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic means. One method of separating the nucleic acid strands involves heating the nucleic acid until it is predominately denatured (e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured). The heating conditions necessary for denaturing template nucleic acid will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90° C. to about 105° C. for a time depending on features of the reaction such as temperature and the nucleic acid length. Denaturation is typically performed for about 5 sec to 9 min. In order to not expose the respective polymerase like e.g. the Z05 DNA Polymerase to such high temperatures for too long and thus risking a loss of functional enzyme, it may be desirable to use short denaturation steps.

In an embodiment of the invention, the denaturation step is up to 30 sec, up to 20 sec, up to 10 sec, up to 5 sec, or about 5 sec.

If the double-stranded template nucleic acid is denatured by heat, the reaction mixture is allowed to cool to a temperature that promotes annealing of each primer to its target sequence on the target nucleic acids.

The temperature for annealing can be from about 35° C. to about 70° C., about 45° C. to about 65° C.; about 50° C. to about 60° C., or about 55° C. to about 58° C. Annealing times can be from about 10 sec to about 1 min (e.g., about 20 sec to about 50 sec; about 30 sec to about 40 sec). In this context, it can be advantageous to use different annealing temperatures in order to increase the inclusivity of the respective assay. In brief, this means that at relatively low annealing temperatures, primers may also bind to targets having single mismatches, so variants of certain sequences can also be amplified. This can be desirable if e.g. a certain organism has known or unknown genetic variants which should also be detected. On the other hand, relatively high annealing temperatures bear the advantage of providing higher specificity, since towards higher temperatures the probability of primer binding to not exactly matching target sequences continuously decreases. In order to benefit from both phenomena, in some embodiments of the invention can be desirable that the process described above comprises annealing at different temperatures, for example first at a lower, then at a higher temperature. If, e.g., a first incubation takes place at 55° C. for about 5 cycles, non-exactly matching target sequences may be (pre-)amplified. This can be followed e.g. by about 45 cycles at 58° C., providing for higher specificity throughout the major part of the experiment. This way, potentially important genetic variants are not missed, while the specificity remains relatively high.

The reaction mixture is then adjusted to a temperature at which the activity of the polymerase is promoted or optimized, i.e., a temperature sufficient for extension to occur from the annealed primer to generate products complementary to the nucleic acid to be analyzed. The temperature should be sufficient to synthesize an extension product from each primer that is annealed to a nucleic acid template, but should not be so high as to denature an extension product from its complementary template (e.g., the temperature for extension generally ranges from about 40° to 80° C. (e.g., about 50° C. to about 70° C.; about 60° C.). Extension times can be from about 10 sec to about 5 min, about 15 sec to 2 min, about 20 sec to about 1 min, or about 25 sec to about 35 sec. The newly synthesized strands form a double-stranded molecule that can be used in the succeeding steps of the reaction. The steps of strand separation, annealing, and elongation can be repeated as often as needed to produce the desired quantity of amplification products corresponding to the target nucleic acids. The limiting factors in the reaction are the amounts of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are can be repeated at least once. For use in detection, the number of cycling steps will depend, e.g., on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps are required to amplify the target sequence sufficient for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated as many as 40, 60, or even 100 times.

Within the scope of the invention, a PCR can be carried out in which the steps of annealing and extension are performed in the same step (one-step PCR) or, as described above, in separate steps (two-step PCR). Performing annealing and extension together and thus under the same physical and chemical conditions, with a suitable enzyme such as, for example, the Z05 DNA polymerase, bears the advantage of saving the time for an additional step in each cycle, and also abolishing the need for an additional temperature adjustment between annealing and extension. Thus, the one-step PCR reduces the overall complexity of the respective assay.

In general, shorter times for the overall amplification are desired, as the time-to-result is reduced and leads to a possible earlier diagnosis.

Other exemplary nucleic acid amplification methods to be used in the context of the invention comprise the Ligase Chain Reaction (LCR; Wu D. Y. and Wallace R. B., Genomics 4 (1989) 560-69; and Barany F., Proc. Natl. Acad. Sci. USA 88 (1991)189-193); Polymerase Ligase Chain Reaction (Barany F., PCR Methods and Applic. 1 (1991) 5-16); Gap-LCR (WO 90/01069); Repair Chain Reaction (EP 0439182 A2), 3SR (Kwoh D. Y. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177; Guatelli J. C., et al., Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878; WO 92/08808), and NASBA (U.S. Pat. No. 5,130,238). Further, there are strand displacement amplification (SDA), transcription mediated amplification (TMA), and Qbeta-amplification (for a review see e.g. Whelen A. C. and Persing D. H., Annu. Rev. Microbiol. 50 (1996) 349-373; Abramson R. D. and Myers T. W., Curr Opin Biotechnol 4 (1993) 41-47).

The internal control nucleic acid used in the present invention may exhibit the following properties relating to its sequence:

-   -   a melting temperature from 55° C. to 90° C., from 65° C. to 85°         C., from 70° C. to 80° C., or about 75° C.     -   a length of up to 500 bases or base pairs, from 50 to 300 bases         or base pairs, from 100 to 200 bases or base pairs, or about 180         bases or base pairs     -   a GC content from 30% to 70%, from 40% to 60%, or about 50%.

In the context of the invention, a “sequence” is the primary structure of a nucleic acid, i.e. the specific arrangement of the single nucleobases of which the respective nucleic acids consists. It has to be understood that the term “sequence” does not denote a specific type of nucleic acid such as RNA or DNA, but applies to both as well as to other types of nucleic acids such as e.g. PNA or others. Where nucleobases correspond to each other, particularly in the case of uracil (present in RNA) and thymine (present in DNA), these bases can be considered equivalent between RNA and DNA sequences, as well-known in the pertinent art.

Clinically relevant nucleic acids are often DNA which can be derived e.g. from DNA viruses like e.g. Hepatitis B Virus (HBV), Cytomegalovirus (CMV) and others, or bacteria like e.g. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and others. In such cases, it can be advantageous to use an internal control nucleic acid and/or an external control nucleic acid consisting of DNA, in order to reflect the target nucleic acids properties.

Therefore, an aspect of the invention is the method described above, wherein said internal control nucleic acid and/or said external control nucleic acid in an aqueous buffer are DNA.

On the other hand, numerous nucleic acids relevant for clinical diagnostics are ribonucleic acids, like e.g. the nucleic acids from RNA viruses such as for example Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), the West Nile Virus (WNV), Human Papilloma Virus (HPV), Japanese Encephalitis Virus (JEV), St. Louis Encephalitis Virus (SLEV) and others. The present invention can be readily applied to such nucleic acids. In this case, it can be advantageous to use an internal control nucleic acid and/or an external control nucleic acid consisting of RNA, in order to reflect the target nucleic acids properties. If both RNA and DNA are to be analyzed in the process described supra, for example the internal and/or the external control nucleic acid are RNA, as a control nucleic acid can mimics the most sensitive target of an assay involving multiple targets, and RNA targets usually have to be more closely controlled.

Thus, an aspect of the invention is the method described above, wherein said internal control nucleic acid and/or said external control nucleic acid in an aqueous buffer are RNA.

Since RNA is more prone to degradation than DNA due to influences such as alkaline pH, ribonucleases etc., internal control nucleic acids made of RNA can be provided as armored particles. Armored particles such as especially armored RNA are described e.g. in EP910643. In brief, the RNA, which can be produced chemically or, heterologously e.g. by bacteria such as e.g. E. coli, is at least partially encapsulated in a viral coat protein. The latter confers resistance of the RNA towards external influences, in particular ribonucleases. It must be understood that internal control DNA can also be provided as an armored particle. Typically, armored RNA and/or DNA are obtained by using bacteriophages. In the case of DNA, the Lambda phage is useful in this context, as known to the skilled person. Both armored RNA and DNA are useful as internal and/or external control nucleic acids in the context of the invention

Therefore, an aspect of the invention is the method described above, wherein said internal control nucleic acid and/or said external control nucleic acid is an armored nucleic acid.

In other embodiments of the invention, plasmids may be used as internal and/or external control nucleic acids. Plasmids are circular nucleic acids mostly derived from extragenomic bacterial DNA. Their use in uncoated form as control nucleic acids is known to the person skilled in the art. Such embodiments are particularly useful in connection with the external nucleic acid in an aqueous buffer as used in the invention, since in an aqueous buffer the external control nucleic acid need not be as carefully protected against adverse influences such as nucleases, as would be the case in e.g. NHP, since the latter can exhibit significant nuclease activity.

Hence, an aspect of the invention is the method described above, wherein said external control nucleic acid is a plasmid. For example, said plasmid is uncoated. Further, said plasmid is DNA.

Typically, in amplification-based nucleic acid diagnostics, RNA templates are transcribed into DNA prior to amplification and detection.

Hence, an aspect of the invention is the process described above, wherein said amplification reagents comprise a polymerase with reverse transcriptase activity, said process further comprising between step e. and step f. the step of incubating in said reaction vessels said purified nucleic acids with said one or more amplification reagents for a period of time and under conditions suitable for transcription of RNA by said polymerase with reverse transcriptase activity to occur.

A “polymerase with reverse transcriptase activity” is a nucleic acid polymerase capable of synthesizing DNA based on an RNA template. It is also capable of the formation of a double-stranded DNA once the RNA has been reverse transcribed into a single strand cDNA. In an embodiment of the invention, the polymerase with reverse transcriptase activity is thermostable.

In an embodiment, the process according to the invention comprises incubating a sample containing an RNA template with an oligonucleotide primer sufficiently complementary to said RNA template to hybridize with the latter, and a thermostable DNA polymerase in the presence of at least all four natural or modified deoxyribonucleoside triphosphates, in an appropriate buffer comprising a metal ion buffer which, in an embodiment, buffers both the pH and the metal ion concentration. This incubation is performed at a temperature sufficient for said primer to hybridize to said RNA template and said DNA polymerase to catalyze the polymerization of said deoxyribonucleoside triphosphates to form a cDNA sequence complementary to the sequence of said RNA template.

As used herein, the term “cDNA” refers to a complementary DNA molecule synthesized using a ribonucleic acid strand (RNA) as a template. The RNA may e.g. be mRNA, tRNA, rRNA, or another form of RNA, such as viral RNA. The cDNA may be single-stranded, double-stranded or may be hydrogen-bonded to a complementary RNA molecule as in an RNA/cDNA hybrid.

A primer suitable for annealing to an RNA template may also be suitable for amplification by PCR. For PCR, a second primer, complementary to the reverse transcribed cDNA strand, provides an initiation site for the synthesis of an extension product.

In the amplification of an RNA molecule by a DNA polymerase, the first extension reaction is reverse transcription using an RNA template, and a DNA strand is produced. The second extension reaction, using the DNA template, produces a double-stranded DNA molecule. Thus, synthesis of a complementary DNA strand from an RNA template by a DNA polymerase provides the starting material for amplification.

Thermostable DNA polymerases can be used in a coupled, one-enzyme reverse transcription/amplification reaction. The term “homogeneous”, in this context, refers to a two-step single addition reaction for reverse transcription and amplification of an RNA target. By homogeneous it is meant that following the reverse transcription (RT) step, there is no need to open the reaction vessel or otherwise adjust reaction components prior to the amplification step. In a non-homogeneous RT/PCR reaction, following reverse transcription and prior to amplification one or more of the reaction components such as the amplification reagents are e.g. adjusted, added, or diluted, for which the reaction vessel has to be opened, or at least its contents have to be manipulated. While both homogeneous and non-homogeneous embodiments are comprised by the scope of the invention, the homogeneous format for RT/PCR is desirable.

Reverse transcription is an important step in an RT/PCR. It is, for example, known in the art that RNA templates show a tendency towards the formation of secondary structures that may hamper primer binding and/or elongation of the cDNA strand by the respective reverse transcriptase. Thus, relatively high temperatures for an RT reaction are advantageous with respect to efficiency of the transcription. On the other hand, raising the incubation temperature also implies higher specificity, i.e. the RT primers will not anneal to sequences that exhibit mismatches to the expected sequence or sequences. Particularly in the case of multiple different target RNAs, it can be desirable to also transcribe and subsequently amplify and detect sequences with single mismatches, e.g. in the case of the possible presence of unknown or rare substrains or subspecies of organisms in the fluid sample.

In order to benefit from both advantages described above, i.e. the reduction of secondary structures and the reverse transcription of templates with mismatches, it is possible to carry out the RT incubation at more than one different temperature.

Therefore, an aspect of the invention is the process described above, wherein said incubation of the polymerase with reverse transcriptase activity is carried out at different temperatures from 30° C. to 75° C., from 45° C. to 70° C., or from 55° C. to 65° C.

As a further important aspect of reverse transcription, long RT steps can damage the DNA templates that may be present in the fluid sample. If the fluid sample contains both RNA and DNA species, it is thus favorable to keep the duration of the RT steps as short as possible, but at the same time ensuring the synthesis of sufficient amounts of cDNA for the subsequent amplification and optional detection of amplificates.

Thus, an aspect of the invention is the process described above, wherein the period of time for incubation of the polymerase with reverse transcriptase activity is up to 30 minutes, 20 minutes, 15 minutes, 12.5 minutes, 10 minutes, 5 minutes, or 1 minute.

A further aspect of the invention is the process described above, wherein the polymerase with reverse transcriptase activity and comprising a mutation is selected from the group consisting of

-   -   a. a CS5 DNA polymerase     -   b. a CS6 DNA polymerase     -   c. a Thermotoga maritima DNA polymerase     -   d. a Thermus aquaticus DNA polymerase     -   e. a Thermus thermophilus DNA polymerase     -   f. a Thermus flavus DNA polymerase     -   g. a Thermus filiformis DNA polymerase     -   h. a Thermus sp. sps17 DNA polymerase     -   i. a Thermus sp. Z05 DNA polymerase     -   j. a Thermotoga neapolitana DNA polymerase     -   k. a Termosipho africanus DNA polymerase     -   l. a Thermus caldophilus DNA polymerase

Particularly suitable for these requirements are enzymes carrying a mutation in the polymerase domain that enhances their reverse transcription efficiency in terms of a faster extension rate.

Therefore, an aspect of the invention is the process described above, wherein the polymerase with reverse transcriptase activity is a polymerase comprising a mutation conferring an improved nucleic acid extension rate and/or an improved reverse transcriptase activity relative to the respective wildtype polymerase.

In an embodiment, in the process described above, the polymerase with reverse transcriptase activity is a polymerase comprising a mutation conferring an improved reverse transcriptase activity relative to the respective wildtype polymerase.

Polymerases carrying point mutations that render them particularly useful in the context of the invention are disclosed in WO 2008/046612. In particular, it may be desirable that polymerases to be used in the context of the present invention are mutated DNA polymerases comprising at least the following motif in the polymerase domain:

T-G-R-L-S—S—X_(b7)—X_(b8)—P—N-L-Q-N; wherein X_(b7) is an amino acid selected from S or T and wherein X_(b8) is an amino acid selected from G, T, R, K, or L, wherein the polymerase comprises 3′-5′ exonuclease activity and has an improved nucleic acid extension rate and/or an improved reverse transcription efficiency relative to the wildtype DNA polymerase, wherein in said wildtype DNA polymerase X_(b8) is an amino acid selected from D, E or N.

One example is mutants of the thermostable DNA polymerase from Thermus species Z05 (described e.g. in U.S. Pat. No. 5,455,170), said mutants comprising mutations in the polymerase domain as compared with the respective wildtype enzyme Z05. An exemplary polymerase for the method according to the invention is a mutant Z05 DNA polymerase wherein the amino acid at position 580 is selected from the group consisting of G, T, R, K and L.

For reverse transcription using a thermostable polymerase, Mn2+ is can be used as the divalent cation and is typically included as a salt, for example, manganese chloride (MnCl2), manganese acetate (Mn(OAc)₂), or manganese sulfate (MnSO₄). If MnCl2 is included in a reaction containing 50 mM Tricine buffer, for example, the MnCl2 is generally present at a concentration of 0.5-7.0 mM; 0.8-1.4 mM can be used when 200 mM of each dGTP, dATP, dUTP, and, dCTP are utilized; for example 2.5-3.5 mM MnCl2. Further, the use of Mg2+ as a divalent cation for reverse transcription is also possible in the context of the present invention.

Since it is in the scope of the invention to reverse-transcribe RNA target nucleic acids into cDNA while preserving the DNA target nucleic acids so both cDNA and DNA can be used for subsequent amplification, the internally and externally controlled process described above is particularly useful for the simultaneous amplification of target nucleic acids derived from both organisms having an RNA genome or organisms having a DNA genome. This advantage considerably increases the spectrum of different organisms, especially pathogens, that can be analyzed under identical physical conditions.

Therefore, an aspect of the invention is the process described above, wherein the at least two target nucleic acids comprise RNA and DNA.

“Simultaneously” or “simultaneous”, in the sense of the invention, means that two actions, such as amplifying a first and a second or more nucleic acids, are performed at the same time and under the same physical conditions. In one embodiment, simultaneous amplification of at least a first and a second target nucleic acid is performed in one vessel. In another embodiment, simultaneous amplification is performed with at least one nucleic acid in one vessel and at least a second nucleic acid in a second vessel, at the same time and under the same physical conditions, particularly with respect to temperature and incubation time wherein the internal control nucleic acid mentioned above is present each of said vessels, whereas the external control nucleic acid or acids are present only in a different vessel or vessels.

An “organism”, as used herein, means any living single- or multicellular life form. In the context of the invention, a virus is an organism.

Especially due to an appropriate temperature optimum, enzymes like Tth polymerase or, the mutant Z05 DNA polymerase mentioned above are suited to carry out the subsequent step of amplification of the target nucleic acids. Exploiting the same enzyme for both reverse transcription and amplification contributes to the ease of carrying out the process and facilitates its automation, since the fluid sample does not have to be manipulated between the RT and the amplification step.

Therefore, in an embodiment, in the process described above the same polymerase with reverse transcriptase activity is used for reverse transcription and for the amplification in step f. For example, the enzyme is the mutant Z05 DNA polymerase described supra.

In order not to expose the polymerase or other components of the reaction mixture in the context of the invention, in an embodiment, steps above 90° C. are up to 20 sec, up to 15 sec, up to 10 sec, up to 5 sec or 5 sec long. This also reduces the time-to-result and cuts down the overall required time of the assay.

In such a homogeneous setup, it can be of considerable advantage to seal the reaction vessels prior to initiating the RT and the amplification, thereby reducing the risk of contamination. Sealing can be e.g. achieved by applying a foil that is transparent, a cap, or by oil added to the reaction vessels and forming a lipophilic phase as a sealing layer at the top of the fluid.

Thus, an aspect of the invention is the process described above, further comprising after step e. the step of sealing the at least two reaction vessels.

For the ease of handling and to facilitate automation, it is possible to combine the at least two reaction vessels in an integral arrangement, so they can be manipulated together.

Consequently, an aspect of the invention is the process described above, wherein the at least two reaction vessels are combined in the same integral arrangement.

Integral arrangements can e.g. be vials or tubes reversibly or irreversibly attached to each other or arranged in a rack. For example, the integral arrangement is a multiwell plate.

The target of the amplification step can be an RNA/DNA hybrid molecule. The target can be a single-stranded or double-stranded nucleic acid. Although the most widely used PCR procedure uses a double-stranded target, this is not a necessity. After the first amplification cycle of a single-stranded DNA target, the reaction mixture contains a double-stranded DNA molecule consisting of the single-stranded target and a newly synthesized complementary strand. Similarly, following the first amplification cycle of an RNA/cDNA target, the reaction mixture contains a double-stranded cDNA molecule. At this point, successive cycles of amplification proceed as described above.

Since nucleic acid amplification, especially but not only in the case of PCR, is very efficient if carried out as a cycling reaction, an aspect of the invention is the process described above, wherein the amplification reaction in step f. consists of multiple cycling steps.

Suitable nucleic acid detection methods are known to the expert in the field and are described in standard textbooks as Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 and Ausubel F. et al.: Current Protocols in Molecular Biology 1987, J. Wiley and Sons, NY. There may be also further purification steps before the nucleic acid detection step is carried out such as e.g. a precipitation step. The detection methods may include but are not limited to the binding or intercalating of specific dyes such as ethidium bromide which intercalates into the double-stranded DNA and changes its fluorescence thereafter. The purified nucleic acid may also be separated by electrophoretic methods optionally after a restriction digest and visualized thereafter. There are also probe-based assays which exploit the oligonucleotide hybridization to specific sequences and subsequent detection of the hybrid.

It may be desirable to detect the amplified target nucleic acid during or after the amplification reaction in order to evaluate the result of the analysis. Particularly for detection in real time, it is advantageous to use nucleic acid probes.

Thus, an aspect of the invention is the process described above, wherein a cycling step comprises an amplification step and a hybridization step, said hybridization step comprising hybridizing the amplified nucleic acids with probes.

It can be favorable to monitor the amplification reaction in real time, i.e. to detect the target nucleic acids and/or their amplificates during the amplification itself.

Therefore, an aspect of the invention is the process described above, wherein the probes are labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety.

The methods set out above can be based on Fluorescence Resonance Energy Transfer (FRET) between a donor fluorescent moiety and an acceptor fluorescent moiety. A representative donor fluorescent moiety is fluorescein, and representative corresponding acceptor fluorescent moieties include LC-Red 640, LC-Red 705, Cy5, and Cy5.5. Typically, detection includes exciting the sample at a wavelength absorbed by the donor fluorescent moiety and visualizing and/or measuring the wavelength emitted by the corresponding acceptor fluorescent moiety. In the process according to the invention, detection can be followed by quantitating the FRET. For example, detection is performed after each cycling step. Further for example, detection is performed in real time. By using commercially available real-time PCR instrumentation (e.g., LightCycler or TaqMan), PCR amplification and detection of the amplification product can be combined in a single closed cuvette with dramatically reduced cycling time. Since detection occurs concurrently with amplification, the real-time PCR methods obviate the need for manipulation of the amplification product, and diminish the risk of cross-contamination between amplification products. Real-time PCR greatly reduces turn-around time and is an attractive alternative to conventional PCR techniques in the clinical laboratory.

The following patent applications describe real-time PCR as used in the LightCycler technology: WO 97/46707, WO 97/46714 and WO 97/46712. The LightCycler instrument is a rapid thermal cycler combined with a microvolume fluorometer utilizing high quality optics. This rapid thermocycling technique uses thin glass cuvettes as reaction vessels. Heating and cooling of the reaction chamber are controlled by alternating heated and ambient air. Due to the low mass of air and the high ratio of surface area to volume of the cuvettes, very rapid temperature exchange rates can be achieved within the thermal chamber.

TaqMan technology utilizes a single-stranded hybridization probe labeled with two fluorescent moieties. When a first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to a second fluorescent moiety according to the principles of FRET. The second fluorescent moiety is generally a quencher molecule. Typical fluorescent dyes used in this format are for example, among others, FAM, HEX, CY5, JA270, Cyan and CY5.5. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target nucleic acid (i.e., the amplification product) and is degraded by the 5′ to 3′ exonuclease activity of the Taq or another suitable polymerase as known by the skilled artisan, such as the mutant Z05 polymerase, during the subsequent elongation phase. As a result, the excited fluorescent moiety and the quencher moiety become spatially separated from one another. As a consequence, upon excitation of the first fluorescent moiety in the absence of the quencher, the fluorescence emission from the first fluorescent moiety can be detected.

In both detection formats described above, the intensity of the emitted signal can be correlated with the number of original target nucleic acid molecules.

As an alternative to FRET, an amplification product can be detected using a double-stranded DNA binding dye such as a fluorescent DNA binding dye (e.g., SYBRGREEN I® or SYBRGOLD® (Molecular Probes)). Upon interaction with the double-stranded nucleic acid, such fluorescent DNA binding dyes emit a fluorescence signal after excitation with light at a suitable wavelength. A double-stranded DNA binding dye such as a nucleic acid intercalating dye also can be used. When double-stranded DNA binding dyes are used, a melting curve analysis is usually performed for confirmation of the presence of the amplification product.

Molecular beacons in conjunction with FRET can also be used to detect the presence of an amplification product using the real-time PCR methods of the invention. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and the fluorescent labels are typically located at each end of the probe. Molecular beacon technology uses a probe oligonucleotide having sequences that permit secondary structure formation (e.g. a hairpin). As a result of secondary structure formation within the probe, both fluorescent moieties are in spatial proximity when the probe is in solution. After hybridization to the amplification products, the secondary structure of the probe is disrupted and the fluorescent moieties become separated from one another such that after excitation with light of a suitable wavelength, the emission of the first fluorescent moiety can be detected.

Thus, a method according to the invention is the method described above using FRET, wherein said probes comprise a nucleic acid sequence that permits secondary structure formation, wherein said secondary structure formation results in spatial proximity between said first and second fluorescent moiety.

Efficient FRET can only take place when the fluorescent moieties are in direct local proximity and when the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety.

Thus, in an embodiment, said donor and acceptor fluorescent moieties are within no more than 5 nucleotides of each other on said probe.

In a further embodiment, said acceptor fluorescent moiety is a quencher.

As described above, in the TaqMan format, during the annealing step of the PCR reaction, the labeled hybridization probe binds to the target nucleic acid (i.e., the amplification product) and is degraded by the 5′- to 3′-exonuclease activity of the Taq or another suitable polymerase as known by the skilled artisan, such as the mutant Z05 polymerase, during the subsequent elongation phase.

Thus, in an embodiment, in the process described above, amplification employs a polymerase enzyme having 5′- to 3′-exonuclease activity.

It is further advantageous to carefully select the length of the amplicon that is yielded as a result of the process described above. Generally, relatively short amplicons increase the efficiency of the amplification reaction. Thus, an aspect of the invention is the process described above, wherein the amplified fragments comprise up to 450 bases, up to 300 bases, up to 200 bases, or up to 150 bases.

The internal control nucleic acid and/or the external control nucleic acid used in the present invention can serve as a “quantitative standard nucleic acid” which is apt to be and used as a reference in order to quantify, i.e. to determine the quantity of the target nucleic acids. For this purpose, one or more quantitative standard nucleic acids undergo all possible sample preparation steps along with the target nucleic acids. Moreover, a quantitative standard nucleic acid is processed throughout the method within the same reaction mixture. It must generate, directly or indirectly, a detectable signal both in the presence or absence of the target nucleic acid.

“To generate” means to produce, directly or indirectly. In the context of a “detectable signal”, “to generate” can therefore mean “to produce directly”, e.g. in the case of a fluorescent dye emitting a fluorescent signal, or “to produce indirectly” in the sense of “to evoke” or “to induce”, such as a “microbial nucleic acid” “generating” a “detectable signal” via a “label” such as a “fluorescent dye”, or via a nucleic acid probe carrying a “label” such as a “fluorescent dye”.

The concentration of the quantitative standard nucleic acid has to be carefully optimized in each test in order not to interfere with sensitivity but in order to generate a detectable signal also e.g. at very high target concentrations. In terms of the limit of detection (LOD, see below) of the respective assay, the concentration range for the “quantitative standard nucleic acid” is 20-5000×LOD, 20-1000×LOD, or 20-5000×LOD. The final concentration of the quantitative standard nucleic acid in the reaction mixture is dependent on the quantitative measuring range accomplished.

“Limit of detection” or “LOD” means the lowest detectable amount or concentration of a nucleic acid in a sample. A low “LOD” corresponds to high sensitivity and vice versa. The “LOD” is usually expressed either by means of the unit “cp/ml”, particularly if the nucleic acid is a viral nucleic acid, or as IU/ml. “Cp/ml” means “copies per milliliter” wherein a “copy” is copy of the respective nucleic acid. IU/ml stands for “International units/ml”, referring to the WHO standard.

A widely used method for calculating an LOD is “Probit Analysis”, which is a method of analyzing the relationship between a stimulus (dose) and the quantal (all or nothing) response. In a typical quantal response experiment, groups of animals are given different doses of a drug. The percent dying at each dose level is recorded. These data may then be analyzed using Probit Analysis. The Probit Model assumes that the percent response is related to the log dose as the cumulative normal distribution. That is, the log doses may be used as variables to read the percent dying from the cumulative normal distribution. Using the normal distribution, rather than other probability distributions, influences the predicted response rate at the high and low ends of possible doses, but has little influence near the middle.

The Probit Analysis can be applied at distinct “hitrates”. As known in the art, a “hitrate” is commonly expressed in percent [%] and indicates the percentage of positive results at a specific concentration of an analyte. Thus for example, an LOD can be determined at 95% hitrate, which means that the LOD is calculated for a setting in which 95% of the valid results are positive.

In an embodiment, the process described above provides an LOD of 1 to 100 cp/ml or 0.5 to 50 IU/ml, of 1 to 75 cp/ml or 0.5 to 30 IU/ml, of 1 to 25 cp/ml or 1 to 20 IU/ml.

With respect to some examples of possible target nucleic acids from certain viruses, the process described above provides the following LODs:

-   -   HIV: up to 60 cp/ml, up to 50 cp/ml, up to 40 cp/ml, up to 30         cp/ml, up to 20 cp/ml, or up to 15 cp/ml     -   HBV: up to 10 IU/ml, up to 7.5 IU/ml, or up to 5 IU/ml     -   HCV: up to 10 IU/ml, up to 7.5 IU/ml, or up to 5 IU/ml     -   WNV I: up to 20 cp/ml, up to 15 cp/ml, or up to 10 cp/ml     -   WNV II: up to 20 cp/ml, up to 15 cp/ml, up to 10 cp/ml, or up to         5 cp/ml     -   JEV: up to 100 cp/ml, up to 75 cp/ml, up to 50 cp/ml, or up to         30 cp/ml     -   SLEV: up to 100 cp/ml, up to 75 cp/ml, up to 50 cp/ml, up to 25         cp/ml, or up to 10 cp/ml.

An example of how to perform calculation of quantitative results in the TaqMan format based on an internal control nucleic acid serving as a quantitative standard nucleic acid is described in the following: A titer is calculated from input data of instrument-corrected fluorescence values from an entire PCR run. A set of samples containing a target nucleic acid and an internal control nucleic acid serving as a quantitative standard nucleic acid undergo PCR on a thermocycler using a specified temperature profile. At selected temperatures and times during the PCR profile samples are illuminated by filtered light and the filtered fluorescence data are collected for each sample for the target nucleic acid and the internal control nucleic acid. After a PCR run is complete, the fluorescence readings are processed to yield one set of dye concentration data for the internal control nucleic acid and one set of dye concentration data for the target nucleic acid. Each set of dye concentration data is processed in the same manner. After several plausibility checks, the elbow values (CT) are calculated for the internal control nucleic acid and the target nucleic acid. The elbow value is defined as the point where the fluorescence of the target nucleic acid or the internal control nucleic acid crosses a predefined threshold (fluorescence concentration). Titer determination is based on the assumptions that the target nucleic acid and the internal and/or external control nucleic acid are amplified with the same efficiency and that at the calculated elbow value equal amounts of amplicon copies of target nucleic acid and internal control nucleic acid are amplified and detected. Therefore, the (CTQS−CTtarget) is linear to log(target conc/QS conc). In this context, QS denotes the quantitative standard nucleic acid. The titer T can then be calculated for instance by using a polynomial calibration formula as in the following equation: T′=10(a(CTQS−CTtarget)² +b(CTQS−CTtarget)+c)

The polynomial constants and the concentration of the quantitative standard nucleic acid are known, therefore the only variable in the equation is the difference (CTQS−CTtarget).

Further, in the sense of the invention, the internal control nucleic acid or the external control nucleic acid can serve as a “qualitative control nucleic acid”. A “qualitative control nucleic acid” is particularly useful for confirming the validity of the test result of a qualitative detection assay: Even in the case of a negative result, the qualitative control must be detected, otherwise the test itself is considered to be inoperative. However, in a qualitative setup, it does not necessarily have to be detected in case of a positive result. As a consequence, its concentration must be relatively low. It has to be carefully adapted to the respective assay and its sensitivity. For example, the concentration range for the qualitative control nucleic acid comprises a range of 1 copy per reaction to 1000 copies per reaction. In relation to the respective assay's limit of detection (LOD), its concentration is between the LOD of an assay and the 25 fold value of the LOD, or between the LOD and 10×LOD. Or, it is between 2× and 10×LOD, or between 5× and 10×LOD. For example, it is 5× or 10×LOD.

The results described above may be adulterated and, for example, comprise false-positives, in the case of cross-contamination with nucleic acids from sources other than the fluid sample. In particular, amplificates of former experiments may contribute to such undesired effects. One particular method for minimizing the effects of cross-contamination of nucleic acid amplification is described in U.S. Pat. No. 5,035,996. The method involves the introduction of unconventional nucleotide bases, such as dUTP, into the amplified product and exposing carryover products to enzymatic and/or physicochemical treatment to render the product DNA incapable of serving as a template for subsequent amplifications. Enzymes for such treatments are known in the art. For example, uracil-DNA glycosylase, also known as uracil-N-glycosylase or UNG, removes uracil residues from PCR products containing that base. The enzyme treatment results in degradation of the contaminating carryover PCR product and thus serves to decontaminate the amplification reaction.

Thus, an aspect of the invention is the process described above, further comprising between step d. and step e. the steps of

-   -   treating the fluid sample with an enzyme under conditions in         which products from amplifications of cross-contaminating         nucleic acids from other samples are enzymatically degraded;     -   inactivating said enzyme.

For example, the enzyme is uracil-N-glycosylase.

In the process according to the invention, all steps can be automated. “Automated” means that the steps of a process are suitable to be carried out with an apparatus or machine capable of operating with little or no external control or influence by a human being. Only the preparation steps for the method may have to be done by hand, e.g. storage containers have to be filled and put into place, the choice of samples has to be performed by a human being and further steps known to the expert in the field, e.g. the operation of a controlling computer. The apparatus or machine may e.g. automatically add liquids, mix the samples or carry out incubation steps at specific temperatures. Typically, such a machine or apparatus is a robot controlled by a computer which carries out a program in which the single steps and commands are specified.

A further aspect of the invention is an analytical system (440) for amplifying a target nucleic acid that may be present in at least one fluid sample, said system comprising:

-   -   an amplification station (405) comprising reaction vessels, said         reaction vessels comprising amplification reagents, purified         target nucleic acid and a purified internal control nucleic         acid, and at least one external control nucleic acid in an         aqueous buffer,         wherein said at least one external control nucleic acid in an         aqueous buffer is comprised in a different vessel than said         purified target nucleic acid.

An “analytical system” is an arrangement of components such as instruments interacting with each other with the ultimate aim to analyze a given sample.

The advantages of said analytical system are the same as described supra with respect to the process according to the invention.

The analytical system (440, FIG. 11) of the present invention is a system (440) comprising a module (401) for isolating and/or purifying an analyte. Further, the system (440) additionally comprises a module (403) for analyzing said analyte to obtain a detectable signal. The detectable signal can be detected in the same module (401, 402, 403) or, alternatively, in a separate module. The term “module” as used herein relates to any spatially defined location within the analyzer (400). Two modules (401, 403) can be separated by walls, or can be in open relationship. Any one module (401, 402, 403) can be either autonomously controlled, or control of the module (401, 402, 403) can be shared with other modules. For example, all modules are controlled centrally. Transfer between modules (401, 402, 403) can be manual, or automated. Thus, a number of different embodiments of automated analyzers (400) are encompassed by the present invention.

An aspect of the invention is the analytical system described above, further comprising

-   -   a separation station comprising a solid support material, said         separation station being constructed and arranged to separate         and purify nucleic acids comprised in said at least one fluid         sample.

The “separation station” is described supra.

An “amplification station” comprises a temperature-controlled incubator for incubating the contents of at least two reaction vessels. It further comprises a variety of reaction vessels like tubes or plates, in which a reaction for the analysis of the sample such as PCR takes place. The outer limits or walls of such vessels are chemically inert such that they do not interfere with the amplification reaction taking place within. For the ease of handling and to facilitate automation, it may be desirable to combine the at least two reaction vessels in an integral arrangement, so they can be manipulated together.

Consequently, an aspect of the invention is the analytical system described above, wherein the at least two reaction vessels are combined in an integral arrangement.

Integral arrangements can e.g. be vials or tubes reversibly or irreversibly attached to each other or arranged in a rack. For example, the integral arrangement is a multiwell plate.

Hence, an aspect of the invention is the analytical system described above, wherein said integral arrangement is a multiwell plate and said at least one external control nucleic acid in an aqueous buffer is comprised in a different well than said purified target nucleic acid.

As described in the context of the process according to the invention, in some embodiments a negative control is added as defined supra.

Therefore, an aspect of the invention is the analytical system described above, wherein said amplification station further comprises a negative control in a different vessel than said purified target nucleic acid and said external control nucleic acid in an aqueous buffer.

In a further aspect of the invention, said negative control is the same buffer that serves as a fluid matrix for said external control nucleic acid.

In another embodiment, said negative control is water.

For example, said multiwell plate is held in a holding station. In an embodiment, one handler transports a multiwell vessel from a holding station to an air-lock (460), and a second handler transports said multiwell plate from said air-lock to said amplification station, wherein both handlers interact with said multiwell plate by a form-locking interaction.

In an embodiment, the analytical system is fully automated.

In one embodiment, at least two reaction vessels combined in an integral arrangement are transported between stations of the system.

In a second embodiment, the purified target nucleic acid is transferred from said separation station to said amplification station. For example, a pipettor comprising pipets with attached pipet tips transfers the liquid comprising the purified nucleic acid.

In a third embodiment, the purified nucleic acid is transferred from said separation station to a reaction vessel in an integral arrangement held in a holding station. For example, said reaction vessel in an integral arrangement is then transferred from said holding station to said amplification station.

The analytical system according to the invention may further comprise a pipetting unit. Said pipetting unit comprises at least one pipet, for example multiple pipets. In an embodiment, said multiple pipets are combined in one or more integral arrangements, within which the pipets can be manipulated individually. Pipets used in the context of the invention can be pipets comprising pipet tips as described supra. In another embodiment, the pipets are pipetting needles.

Alternatively, a reaction vessel or arrangement of reaction vessels used for sample preparation in the separation station and containing the fluid comprising the purified target nucleic acids may be transferred from the separation station to the amplification station.

For this purpose, the analytical system according to the invention may further comprises a transfer unit, said transfer unit comprising a robotic device, said device for example comprising a handler.

For the reasons set out above in the context of the process according to the invention, the following are further aspects of the invention:

-   -   The analytical system (440) described above wherein at least one         reaction vessel comprises an RNA target nucleic acid and a DNA         target nucleic acid.     -   The analytical system (440) described above, wherein at least         one reaction vessel comprises an RNA target nucleic acid, and at         least one other reaction vessel comprises a DNA target nucleic         acid.

For example, the analytical system (440) described above further comprises one or more elements selected from the group consisting of:

-   -   a detection module (403) for detecting signals evoked by an         analyte     -   a sealer (410)     -   a storage module (1008) for reagents and/or disposables.     -   a control unit (1006) for controlling system components.

A “detection module” (403) can e.g. be an optical detection unit for detecting the result or the effect of the amplification procedure. An optical detection unit may comprise a light source, e.g. a xenon lamp, optics such as mirrors, lenses, optical filters, fiber optics for guiding and filtering the light, one or more reference channels, or a CCD camera or a different camera.

A “sealer” (410) is constructed and arranged to seal any vessels used in connection with the analytical system according to the invention. Such a sealer can, for example, seal tubes with appropriate caps, or multiwell plates with foil, or other suitable sealing materials.

A “storage module” (1008) stores the necessary reagents to bring about a chemical or biological reaction important for analysis of the fluid sample. It can also comprise further components useful for the method of the invention, e.g. disposables such as pipet tips or vessels to be used as reaction vessels within the separation station and/or the amplification station.

For example, the analytical system according to the invention further comprises a control unit for controlling system components.

Such a “control unit” (1006) may comprise software for ensuring that the different components of said analytical system work and interact correctly and with the correct timing, e.g. moving and manipulating components such as pipets in a coordinated manner. The control unit may also comprise a processor running a real-time operating system (RTOS), which is a multi-tasking operating system intended for real-time applications. In other words the system processor is capable of managing real-time constraints, i.e. operational deadlines from event to system response regardless of system load. It controls in real time that different units within the system operate and respond correctly according to given instructions.

In an embodiment, the present invention relates to an analytical system (440) for processing an analyte, comprising

a. a first position comprising first receptacles (1001) in linear arrangement comprising liquid samples (1010), a processing plate (101) comprising receptacles (103) in n×m arrangement for holding a liquid sample (1011), a first pipetting device (700) comprising at least two pipetting units (702) in linear arrangement, wherein said pipetting units (702) are coupled to pipette tips (3, 4), and a tip rack (70) comprising pipette tips (3, 4) in an a×(n×m) arrangement; b. a second position comprising a holder (201, 128) for said processing plate (101), a holder (330) for a multiwell plate, a holder (470) for said tip rack (70) and a second pipetting device (35), said second pipetting device (35) comprising pipetting units (702) in an n×m arrangement for coupling to pipette tips (3, 4) (FIG. 12). The term “holder” as used herein relates to any arrangement capable of receiving a rack or a processing plate.

The advantages of the analytical system (440) of the present invention are as described above for the method of the present invention.

For example, the position of said pipetting units (702) of the first pipetting device (700) are variable. Other embodiments of said first pipetting device (700) are described hereinafter.

In one embodiment, the tip rack (70) comprises pipette tips (3, 4) in an a×(n×m) arrangement. For example, a first type (4) and a second type (3) of pipette tips are comprised in the tip rack (70). In this embodiment, the first type of pipette tips (4) is arranged in an n×m arrangement, and the second type of pipette tips (3) is arranged in the n×m arrangement. In this context, “n” denotes the number of rows and m the number of columns, wherein n may be 6, or 8. Further, the first type of pipette tips (4) has a different volume than the second type of pipette tips (3), or, the volume of the first type of pipette tips (4) is more than 500 ul, and the volume of the second type of pipette tips (3) is less than 500 ul. In this embodiment, a=2. However, embodiments of the invention with more than two types of pipette tips, and thus a>2 are also included in the present invention.

In one aspect, the analytical system (440) of the present invention comprises a control unit (1006) for allocating sample types and individual tests to individual positions of said processing plate (101). For example, said positions are separate cells (401, 402).

In one aspect of the invention, the system additionally comprises a transfer system (480) for transferring said process plate (101) and said rack (70) between first (402) and second (401) positions. Other embodiments of said transfer system (480) are conveyor belts or, one or more handler.

Furthermore, said pipette units of said second pipetting device (35) may be are engaged to pipette tips (3, 4) which were used in the first position (402).

An embodiment of the system (440) of the present invention additionally comprises a third station (403) comprising a temperature-controlled incubator for incubating said analyte with reagents necessary to obtain a detectable signal. Further embodiments of this system are described hereinafter.

More optimal control of the allocation of samples and tests to the n×m arrangement is achieved with a first processor (1004) which is comprised in said first position (402) to which said control unit (1006) transfers instructions for allocating sample types and individual tests to specific positions in the n×m arrangement of vessels (103) of the process plate (101), and a second processor (1005) which is comprised in said second position (401) to which said control unit (1006) transfers instructions for allocating sample types and individual tests to specific positions in the n×m arrangement of vessels (103) of the process plate.

For example, said system additionally comprises a first processor located in said first position, and a second processor located in said second position.

Additionally for example, said first processor (1004) controls said first pipetting device (700) and said second processor (1005) controls said second pipetting device (35).

A further aspect of the invention is the process or the analytical system described above comprising more than one external control nucleic acid.

In such embodiments, a plurality of different target nucleic acids possibly contained in single fluid sample or a plurality of different fluid samples can be advantageously analyzed. For instance, said external control nucleic acids can be designed as target-specific control nucleic acids having a similar or identical sequence as the respective target nucleic acid. Methods to design a target-specific external control nucleic acid are known to the person skilled in the art.

All other exemplary embodiments and specific descriptions of embodiments of the analytical system according to the invention are those mentioned for the process according to the invention.

DESCRIPTION OF THE FIGURES

FIG. 1:

Schematic depiction of the sample preparation workflow as used in an embodiment of the invention.

Arrows pointing down denote addition of a component or reagent to each respective well of the deepwell plate mentioned above, arrows pointing up their respective removal. These actions were performed manually in steps 2, 3, 4, 21 and 22, by the process head of the apparatus in steps 10, 14, 16, 18, and 24, and by the reagent head of the apparatus in steps 5, 6, 7, 11, 15 and 19.

It has to be understood that the volumes used can be adjusted flexibly within the spirit of the invention, for example at least about up to 30% of the disclosed values. In particular, in the case of step 2, the sample volume can be variable in order to take into account the different types of fluid samples which may require more or less starting material for obtaining proper results, as known by the artisan. For example, the range is from about 100 ul to about 850 ul. Or, it is about 100 ul, about 500 ul or about 850 ul. Further for example, the volume in the respective vessels is adjusted to an identical total volume with the diluent in step 3. For example, as in the scheme shown in FIG. 1, the total volume adds up to about 850 ul.

FIG. 2 a:

PCR growth curve for HIV in NHP

FIG. 2 b:

PCR growth curve for HIV in aqueous buffer

FIG. 2 c:

PCR growth curve for HCV in NHP

FIG. 2 d:

PCR growth curve for HCV in aqueous buffer

FIG. 2 e:

PCR growth curve for HBV in NHP

FIG. 2 f:

PCR growth curve for HBV in aqueous buffer

FIG. 3:

Perspective view of the processing plate.

FIG. 4:

Perspective view of the processing plate from the opposite angle.

FIG. 5:

Top view of the processing plate.

FIG. 6:

Cross-sectional view along the longer side of the processing plate.

FIG. 7:

A partial view of the cross-sectional view.

FIG. 8:

Perspective view of the longer side of the processing plate.

FIG. 9:

a) to d) show different views of the second embodiment of the magnetic separation station.

FIG. 10:

(a) to (c) show a view of the first embodiment of the magnetic separation station holding the Processing plate, with the first type of magnets in the uppermost Z-position, and the second type of magnets in the lowermost Z-position.

FIG. 11:

Schematic drawings of an analyzer comprising different stations, modules or cells.

FIG. 12:

Shows an analytical system of the present invention.

EXAMPLES

The following examples are offered to illustrate, but not to limit the claimed invention.

The following examples describe an embodiment in which the invention can be worked. It is clear to the person skilled in the art that these examples are not limiting and can be modified without leaving the spirit of the invention.

Example 1

This example describes a process for isolating, amplifying and detecting a target nucleic acid, said process involving an external control nucleic acid in an aqueous buffer.

In brief, in the depicted embodiment, realtime PCR was carried out under identical conditions on the three different viral target nucleic acids HIV, HBV and HCV. For all targets, standard material was used. Suitable standards or other types of targets are available to the skilled artisan.

The instruments listed in the following table were used according to the instructions of the respective manufacturer:

Instrument Manufacturer Hamilton Star Hamilton Medical AG (Bonaduz, CH) Light Cycler 480 Roche Diagnostics GmbH (Mannheim, DE) Chameleon Sealer K biosystems (Essex, UK) Compressor K biosystems (Essex, UK)

A combined panel of HIV, HBV and HCV, with concentrations listed in Table 1, was made in an NHP matrix and in the aqueous buffer matrix according to Table 2. The NHP based panel and the buffer based panel were run in replicates of 28 on the Hamilton Star Proc. 1.6.1. The NHP was acquired from SeraCare Life Science (Milford, Mass., USA).

TABLE 1 Concentration of combined panel HIV HCV HBV Analyte (cp/ml) (cp/ml) (cp/ml) Concentration 350 150 80

TABLE 2 Aqueous buffer IC/IQS—Storage Buffer Conc. or pH Tris (mM) 10 EDTA (mM) 0.1 Sodium Azide (w/v, %) 0.05 Poly(rA) RNA (mg/l) 20 pH 8

For all assays, an RNA molecule serving as a generic internal control nucleic acid was added (100 armored particles/sample). The sequence of said generic control nucleic acid was identical in all cases and selected from the group of SEQ ID NOs 86-89.

Sample preparation was performed on a Hamilton Star (Hamilton, Bonaduz, CH), following the workflow according to the scheme depicted in FIG. 1.

After the final step, the process head of the Hamilton Star apparatus added the respective mastermix (Mmx) containing amplification reagents to each well, mixed the fluids containing the isolated nucleic acids with the Mmx and transferred each resulting mixture to a corresponding well of a microwell plate in which the amplification was carried out.

The following PCR Mmx (consisting of the two reagents R1 and R2) was used for all tested nucleic acids:

TABLE 3 PCR mastermix Final Concentration in 50 ul-PCR (uM) R1 Reagent Mn(Ac)₂ * 4H₂O (pH 6.1 adjusted with Acetic Acid)  3′300 NaN3, buffered with 10 mM Tris at pH7 0.018 R2 Reagent DMSO (%) 5.4 NaN3, buffered with 10 mM Tris at pH7 0.027 K acetate pH 7.0 120′000 Glycerol (%) 3 Tween 20 (%) 0.015 Tricine pH 8.0  60′000 NTQ21-46A—Aptamer 0.2222 Uracil-N-Glycosylase (U/uL) 0.2 dGTP 400.0 dATP 400.0 dCTP 400.0 dUTP 800.0 Z05-D Polymerase (U/ul) 0.9 HIV primers/probes selected from SEQ ID NOs 1-35 0.1-0.3 HBV primers/probes selected from SEQ ID NOs 36-38 0.1-0.3 HCV primers/probes selected from SEQ ID NOs 39-82 0.1-0.3 Control primers/probes selected from SEQ ID NOs 83-85 0.1-0.3 adjust pH to 8.1

The Mmx reagents were combined as follows for the PCR, adding up to a total volume of 50 ul per PCR:

TABLE 4 Composition of PCR reaction mixture R1 10 ul R2 15 ul Eluate (containing isolated nucleic acids) 25 ul

For amplification and detection, the microwell plate was sealed with an automated plate sealer (see above), and the plate was transferred to a LightCycler 480 (see above).

The following PCR profile was used:

TABLE 5 PCR profile Thermo cycling profile Ramp Program Target Acquisition Hold Rate Name (° C.) Mode (hh:mm:ss) (° C./s) Cycles Pre-PCR 50 None 00:02:00 4.4 1 94 None 00:00:05 4.4 55 None 00:02:00 2.2 60 None 00:06:00 4.4 65 None 00:04:00 4.4 1st 95 None 00:00:05 4.4 5 Measurement 55 Single 00:00:30 2.2 2nd 91 None 00:00:05 4.4 45 Measurment 58 Single 00:00:25 2.2 Cooling 40 None 00:02:00 2.2 1

As a result of the assays for all the samples comprised on the microwell plate mentioned above, amplification and detection was achieved in all samples, as depicted in FIG. 2. This shows that the sample preparation prior to amplification was also successfully carried out.

Moreover, the PCR involving nucleic acids in an aqueous buffer (IC buffer) exhibited a comparable efficiency as the PCR involving nucleic acids in normal human plasma (NHP). In detail, the following results were obtained (the corresponding PCR curves are depicted in FIG. 2a-f ):

TABLE 6 PCR results for HIV HIV Channel -2- Sample Hit Comment rate CT RFI IC buffer 28 of average 38.38 8.09 28 STDEV 0.40 0.73 CV 1.05% 8.98% NHP 28 of average 38.17 6.73 28 STDEV 0.48 1.03 CV 1.25% 15.23%

TABLE 7 PCR results for HCV HCV Channel -4- Sample Hit Comment rate CT RFI IC buffer 28 of average 35.51 19.41 28 STDEV 0.22 1.32 CV 0.63% 6.81% NHP 28 of average 35.15 17.30 28 STDEV 0.22 1.18 CV 0.62% 6.83%

TABLE 8 PCR results for HBV HBV Channel -3- Sample Hit Comment rate CT RFI IC buffer with 28 of average 28.10 8.81 PolyRNA 28 STDEV 0.13 0.34 CV 0.45% 3.86% NHP 28 of average 27.43 9.04 28 STDEV 0.18 0.42 CV 0.66% 4.60%

Example 2

The same panel of target material as in Example 1 was assayed under conditions equivalent to the ones previously described.

The target nucleic acids had the following concentrations in the fluid sample:

TABLE 9 Concentration of combined panel HIV HCV HBV Analyte (cp/ml) (cp/ml) (cp/ml) Concentration 150 60 19

In addition to this panel, negative controls (sample matrix not containing any nucleic acid) were assayed.

The following results were obtained:

TABLE 10 Matrix Equivalency Positive Equivalency HIV-1M Ch 2 (FAM) HBV Ch 3 (HEX) HCV Ch 4 (JA270) Hit Hit Hit Sample Rate CT RFI Rate CT RFI Rate CT RFI NHP 84/84 39.55 7.98 84/84 36.14 5.44 84/84 36.94 15.49 (100%) (100%) (100%) IC Buffer 84/84 38.87 8.50 84/84 35.92 5.39 84/84 37.90 14.84 (100%) (100%) (100%) Negative  0/12 — —  0/12 — —  0/12 — — Control  (0%)  (0%)  (0%) NHP Negative  0/12 — —  0/12 — —  0/12 — — Control  (0%)  (0%)  (0%) IC Buffer

Example 3

Under equivalent conditions, the panel of Example 2 was further analyzed in different dilutions as set out in the tables below, in order to determine the LOD (Limit Of Detection) values:

TABLE 11 Sensitivity in NHP - LOD for HIV-1 M HIV-1 M (co-formulated) in NHP Number of Number of Concentration replicates positives Hit rate 40 IU/mL 21 21  100% 20 IU/mL 21 15 71.4% 10 IU/mL 21 15 71.4%  5 IU/mL 21 12 57.1% 2.5 IU/mL  21 4 19.1%  0 IU/mL 21 0   0% LOD by PROBIT analysis (95% hitrate): 41.7 IU/rnL 95% confidence interval for LOD 24.2-128.0 IU/mL by PROBIT analysis:

TABLE 12 Sensitivity in NHP - LOD for HBV HBV (co-formulated) in NHP Number of Number of Concentration replicates positives Hit rate 9.2 IU/mL 21 21 100% 4.6 IU/mL 21 21 100% 2.3 IU/mL 21 21 100% 1.15 IU/mL  21 16 76.2%  0.575 IU/mL  21 13 61.9%    0 IU/mL 21 0  0% LOD by PROBIT analysis (95% hitrate): 1.9 IU/mL 95% confidence interval for LOD 1.3-5.7 IU/mL by PROBIT analysis:

TABLE 13 Sensitivity in NHP - LOD for HCV HCV (co-formulated) in NHP Number of Number of Concentration replicates positives Hit rate 16 IU/mL  21 21 100% 8 IU/mL 21 21 100% 4 IU/mL 21 18 85.7% 2 IU/mL 21 15 71.4% 1 IU/mL 21 12 57.4% 0 IU/mL 21 0   0% LOD by PROBIT analysis (95% hitrate): 5.8 IU/mL 95% confidence interval for LOD 3.7-18.5 IU/mL by PROBIT analysis:

TABLE 14 Sensitivity in IC Buffer - LOD for HIV-1 M HIV-1 M (co-formulated) in IC Buffer Number of Number of Concentration replicates positives Hit rate 40 IU/mL 21 21  100% 20 IU/mL 21 18 85.7% 10 IU/mL 21 16 76.2%  5 IU/mL 21 7 33.3% 2.5 IU/mL  21 4 19.1%  0 IU/mL 21 0   0% LOD by PROBIT analysis (95% hitrate): 27.3 IU/mL 95% confidence interval for LOD 18.2-57.2 IU/mL by PROBIT analysis:

TABLE 15 Sensitivity in IC Buffer - LOD for HBV HBV (co-formulated) in IC Buffer Number of Number of Concentration* replicates positives Hit rate 9.2 IU/mL 21 21 100% 4.6 IU/mL 21 21 100% 2.3 IU/mL 21 21 100% 1.15 IU/mL  21 19 90.5%  0.575 IU/mL  21 15 71.4%    0 IU/mL 21 0  0% LOD by PROBIT analysis (95% hitrate): 1.3 IU/mL 95% confidence interval for LOD 0.9-6.8 IU/mL by PROBIT analysis:

TABLE 16 Sensitivity in IC Buffer - LOD for HCV HCV (co-formulated) in IC Buffer Number of Number of Concentration replicates positives Hit rate 16 IU/mL  21 21  100% 8 IU/mL 21 20 95.2% 4 IU/mL 21 20 95.2% 2 IU/mL 21 14 66.7% 1 IU/mL 21 11 52.4% 0 IU/mL 21 0   0% LOD by PROBIT analysis (95% hitrate): 5.9 IU/mL 95% confidence interval for LOD 3.8-16.9 IU/mL by PROBIT analysis:

TABLE 17 Sensitivity - overview for NHP and IC Buffer LOD by PROBIT analysis (95% hitrate) target NHP IC Buffer HIV-1 M 41.7 IU/mL  27.3 IU/mL  (24.2-128.0 IU/mL) (18.2-57.2 IU/mL) HBV 1.9 IU/mL 1.3 IU/mL (1.3-5.7 IU/mL) (0.9-6.8 IU/mL) HCV 5.8 IU/mL 5.9 IU/mL (3.7-18.5 IU/mL) (3.8-16.9 IU/mL)

Example 4

The HIV standard material described above was quantitatively assayed under conditions equivalent to the ones described supra. The titer of the HIV material was determined in NHP and in the described aqueous buffer (IC Buffer).

HIV was used in the following concentrations (HPC=High Positive Control; LPC=Low Positive Control):

TABLE 18 Titers of quantitated HIV material Process Control Concentration HPC 2E+05 cp/ml LPC 200 cp/ml

This dedicated accuracy study between NHP-based and IC Buffer-based HIV samples demonstrated positive equivalency for both positive controls as evidenced by the mean log 10 titer difference between the two matrices. The mean log 10 titer (cp/mL) difference between the two matrices (NHP—buffer) for the LPC and HPC was:

LPC: −0.03

HPC: −0.08

The target positive controls were tested with 42 replicates per sample matrix.

All mean log 10 titer differences were within ±0.5 log 10 titer. Therefore the experiment shows matrix equivalence for the IC Buffer and the NHP matrix.

Example 5

The LOD values representing the quantitative HIV assay's sensitivity was determined in an analogous manner to the nucleic acid panel described in Example 2. The result demonstrates that the sensitivity of the HIV assay is equivalent in both sample matrices as evidenced by overlapping 95% confidence intervals for both sample matrices (see Table 19 and Table 20).

TABLE 19 Sensitivity in NHP Concentra- Corresponding tion for conc. with Number 100 μL input 500 μL input of rep- Number of volume volume licates positives Hit rate  0 cp/mL  0 cp/mL 21 0 0.0%  30 cp/mL  6 cp/mL 21 15 71.4%  45 cp/mL  9 cp/mL 21 12 57.1% 67.5 cp/mL  13.5 cp/mL   21 17 81.0% 100 cp/mL 20 cp/mL 21 17 81.0% 125 cp/mL 25 cp/mL 21 21 100.0%  150 cp/mL 30 cp/mL 21 20 95.2% Value of PROBIT analysis at 95% hit rate: 34.0 cp/mL 95% confidence interval for value of PROBIT 21.5-154.5 cp/mL analysis at 95% hit rate: ≥95% Hit rate Analysis 25.0 cp/mL

TABLE 20 Sensitivity in IC Buffer Concentra- Corresponding tion for conc. with Number 100 μL input 500 μL input of rep- Number of volume volume licates positives Hit rate  0 cp/mL  0 cp/mL 21 0  0.0%  30 cp/mL  6 cp/mL 21 9 42.9%  45 cp/mL  9 cp/mL 21 13 61.9% 67.5 cp/mL  13.5 cp/mL   21 14 66.7% 100 cp/mL 20 cp/mL 21 19 90.5% 125 cp/mL 25 cp/mL 21 20 95.2% 150 cp/mL 30 cp/mL 21 21 100.0%  Value of PROBIT analysis at 95% hit rate: 25.9 cp/mL 95% confidence interval for value of PROBIT 19.5-45.8 cp/mL analysis at 95% hit rate: ≥95% Hit rate Analysis 25.0 cp/mL

Example 6

A long term stability study for NHP-based and IC Buffer-based HCV samples demonstrated that the stability of the RMC is equivalent in both control matrices as evidenced by the stable hit rate, CT value and RFI value over 6 months storage (see Table 22).

The following high positive (HPC) and low positive controls (LPC) were used:

TABLE 21 Titers of quantitated HCV material Process Control Concentration HPC 1E+05 cp/ml LPC 100 IU/ml

The following results were obtained:

TABLE 22 HCV LPC and HCV HPC stability in NHP and IC Buffer Time CT Stdev RFI Stdev Hit Name Point Value CT Value RFI Rate low PC (NHP) Day 0 35.53 0.34 17.62 1.17 100% 6 months 35.71 0.28 17.26 1.14 100% low PC (IC Day 0 36.08 0.37 20.74 0.71 100% Buffer) 6 months 36.15 0.32 18.95 0.78 100% high PC (NHP) Day 0 23.47 0.16 19.91 1.06 100% 6 months 23.79 0.09 19.89 0.77 100% high PC (IC Day 0 24.25 0.14 23.07 0.52 100% Buffer) 6 months 24.70 0.16 20.63 0.53 100% 

What is claimed is:
 1. A process for controlled amplification of a target nucleic acid that may be present in a fluid sample, comprising: (a) providing in a vessel an internal control nucleic acid in the fluid sample, (b) providing a solid support material to the vessel for a period of time and under conditions sufficient to permit nucleic acids comprising the target nucleic acid and the internal control nucleic acid to be immobilized on the solid support material, (c) isolating the solid support material from other material present in the vessel in a separation station, (d) purifying the nucleic acids in the separation station and washing the solid support material one or more times with a wash buffer, (e) providing (i) in a first reaction vessel the purified nucleic acids of step (d), wherein the purified nucleic acids comprise the internal control nucleic acid and the target nucleic acid derived from the fluid sample, and (ii) in a second reaction vessel an external control nucleic acid in an aqueous buffer, wherein the aqueous buffer lacks a fluid sample, (f) adding identical amplification reagents to each of the first and second reaction vessels, (g) incubating the first and second reaction vessels under identical conditions sufficient for amplification reactions to occur, and (h) detecting results of the amplification reactions, wherein the results of the amplification reactions are indicative of the presence or absence of amplification of the target nucleic acid, the internal control nucleic acid and the external control nucleic acid wherein the aqueous buffer has a pH between 6.0 and 12.0 and comprises: 1-100 mM Tris, 0.01-1 mM EDTA, 0.005-0.5% (w/v) Sodium Azide, and 1-200 mg/l Poly(rA) RNA.
 2. The process of claim 1, wherein the aqueous buffer has a pH of about 8 and comprises: 10 mM Tris, 0.1 mM EDTA, 0.05% (w/v) Sodium Azide, and 20 mg/l Poly(rA) RNA.
 3. The process of claim 1, wherein a negative control is provided in a third reaction vessel and is subjected to steps (b) through (h).
 4. The process of claim 1, wherein the fluid sample is a clinical sample.
 5. The process of claim 1, further comprising: (i) determining the quantity of the target nucleic acid.
 6. The process of claim 1, wherein the fluid sample comprises blood.
 7. The process of claim 1, wherein the fluid sample comprises blood plasma.
 8. The process of claim 1, wherein the aqueous buffer is free of biological buffers derived from blood.
 9. The process of claim 1, wherein the aqueous buffer is free of biological buffers derived from blood plasma.
 10. The process of claim 1, wherein the second reaction vessel comprises more than one external control nucleic acid. 